Multi-arm polymer prodrugs

ABSTRACT

Provided herein are water-soluble prodrugs, compositions comprising such prodrugs, and related methods of making and administering the same. The prodrugs of the invention comprise a water-soluble polymer having three or more arms, at least three of which are typically covalently attached to an active agent, e.g., a small molecule. The conjugates of the invention provide an optimal balance of polymer size and structure for achieving improved drug loading, since the conjugates of the invention possess three or more active agents releasably attached to a multi-armed water-soluble polymer. The prodrugs of the invention are therapeutically effective, and exhibit improved properties in-vivo when compared to unmodified parent drug.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application (i) is a continuation of U.S. patent application Ser.No. 13/765,433, filed Feb. 12, 2013, now U.S. Pat. No. 8,771,662, whichis a continuation of U.S. patent application Ser. No. 11/948,767, filedNov. 30, 2007, now U.S. Pat. No. 8,394,365, which is acontinuation-in-part of U.S. patent application Ser. No. 10/943,799,filed Sep. 17, 2004, now U.S. Pat. No. 7,744,861, which claims thebenefit of priority to U.S. Provisional Application No. 60/503,673,filed Sep. 17, 2003, and to U.S. Provisional Application No, 60/584,308,filed Jun. 30, 2004, and (ii) also claims the benefit of priority toU.S. Provisional Application No. 60/861,995, filed Nov. 30, 2006, and toU.S. Provisional Application No. 61/003,163, filed Nov. 14, 2007, thecontents of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

This invention relates generally to multi-arm, water-soluble polymerdrug conjugates. In particular, the invention is directed to, amongother things, polymer-based prodrugs, pharmaceutical compositionsthereof, and methods for preparing, formulating and administering suchcompositions.

BACKGROUND OF THE INVENTION

Over the years, numerous methods have been proposed for improving thedelivery of biologically active agents. Challenges associated with theformulation and delivery of pharmaceutical agents can include pooraqueous solubility of the pharmaceutical agent, toxicity, lowbioavailability, instability, and rapid in-vivo degradation, to namejust a few. Although many approaches have been devised for improving thedelivery of pharmaceutical agents, no single approach is without itsdrawbacks. For instance, commonly employed drug delivery approachesaimed at solving or at least ameliorating one or more of these problemsinclude drug encapsulation, such as in a liposome, polymer matrix, orunimolecular micelle, covalent attachment to a water-soluble polymersuch as polyethylene glycol, use of gene targeting agents, and the like.

In looking more closely at some of these approaches, liposomeencapsulation is often plagued by low efficiencies of drug loading,resulting in an oftentimes inefficient and cost ineffective process.Moreover, the release rate of the active agent in a liposomalformulation depends upon dissolution or disintegration of the liposome,or diffusion of the active agent through the liposomal layers, therebylimiting the practical availability of the active agent to thebiological system. In addition, liposomal formulations are generallyrestricted to lipid soluble drugs. Polymer matrix-based formulations cansuffer from similar shortcomings, such as the inability towell-characterize such drug delivery systems, particular those that arecross-linked, and the variable release rates associated with activeagents that must diffuse out of a hydrolytically degradable polymermatrix. In comparison, conjugation of an active agent to a polymer suchas polyethylene glycol offers a more well-defined alternative, since theconjugate itself is often although not necessarily well-characterized,particularly in the case of site-specific attachment of the polymer tothe active agent. However, protein-based compositions containingmixtures of positional isomers varying in both the site(s) and number ofpolymer chains attached to a particular protein are not uncommon. Thiscan lead to problems with reproducibly preparing such compositions.

While modification of therapeutic proteins for the purpose of improvingtheir pharmaceutical utility is perhaps one of the most commonapplications of PEGylation, PEGylation has also been used, albeit to alimited degree, to improve the bioavailability and ease of formulationof small molecule therapeutics having poor aqueous solubilities. Forinstance, water-soluble polymers such as PEG have been covalentlyattached to artilinic acid to improve its aqueous solubility (Bentley,et al., U.S. Pat. No. 6,461,603). Similarly, PEG has been covalentlyattached to triazine-based compounds such as trimelamol to improve theirsolubility in water and enhance their chemical stability (Bentley, etal., WO 02/043772). Covalent attachment of PEG to bisindolyl maleimideshas been employed to improve poor bioavailability of such compounds dueto low aqueous solubility (Bentley, et al., WO 03/037384). Prodrugs ofcamptothecin having one or two molecules of camptothecin covalentlyattached to a linear polyethylene glycol have similarly been prepared(Greenwald, et al, U.S. Pat. No. 5,880,131).

Camptothecin (often abbreviated as “CPT”) is a phytotoxic alkaloid firstisolated from the wood and bark of Camptotheca acuminata (Nyssaceae),and has been shown to exhibit antitumor activity. The compound has apentacyclic ring system with an asymmetric center in lactone ring E witha 20 S configuration. The pentacyclic ring system includes apyrrolo[3,4-b]quinoline (rings A, B and C), a conjugated pyridone (ringD), and a six-membered lactone (ring E) with a 20-hydroxyl group. Due toits insolubility in water, camptothecin was initially evaluatedclinically in the form of a water-soluble carboxylate salt having thelactone ring open to form the sodium salt. The sodium salt, althoughexhibiting much improved water solubility in comparison to camptothecinitself, produced severe toxicity and demonstrated very little in vivoanticancer activity, thus demonstrating the undesirability of thisapproach.

It was later discovered that camptothecin and many of its derivativesinhibit topoisomerase, an enzyme that is required for swiveling andrelaxation of DNA during molecular events such as replication andtranscription. Camptothecin stabilizes and forms a reversibleenzyme-camptothecin-DNA ternary complex. The formation of the cleavablecomplex specifically prevents the reunion step of the breakage/unioncycle of the topoisomerase reaction. Topoisomerase I inhibitors are alsoknown to be useful in the treatment of HIV.

In an effort to address the poor aqueous solubility associated withcamptothecin and many of its derivatives, a number of synthetic effortshave been directed to derivatizing the A-ring and/or B-ring oresterifying the 20-hydroxyl to improve water-solubility whilemaintaining cytotoxic activity. For example, topotecan(9-dimethylaminomethyl-10-hydroxy CPT) and irinotecan(7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxy CPT), otherwiseknown as CPT-11, are two water-soluble CPT derivatives that have shownclinically useful activity. Conjugation of certain camptothecinderivatives, such as 10-hydroxycamptothecin and 11-hydroxycamptothecin,to a linear poly(ethylene glycol) molecule via an ester linkage has beendescribed as a means to form water soluble prodrugs (Greenwald, et al.,U.S. Pat. No. 6,011,042). The approach used relies on reaction of anaromatic, hydroxyl-containing compound with an activated polymer.

The clinical effectiveness of many small molecule therapeutics, andoncolytics in particular, is limited by several factors. For instance,irinotecan and other camptothecin derivatives undergo an undesirablehydrolysis of the E-ring lactone under alkaline conditions.Additionally, administration of irinotecan causes a number of troublingside effects, including leucopenia, neutropenia, and diarrhea. Due toits severe diarrheal side-effect, the dose of irinotecan that can beadministered in its conventional, unmodified form is extremely limited,thus hampering the efficacy of this drug and others of this type.

These associated side effects, when severe, can be sufficient to arrestfurther development of such drugs as promising therapeutics. Additionalchallenges facing small molecules include high clearance rates, and inthe instance of anticancer agents, minimal tumor permeation andresidence time. Approaches involving the use of polymer attachment mustbalance the size of the polymer against the molecular weight of theactive agent in order to allow therapeutically effective doses to bedelivered. Finally, the synthesis of a modified or drug-deliveryenhanced active agent must result in reasonable yields, to make any suchapproach economically attractive. Thus, there exists a need for newmethods for effectively delivering drugs, and in particular smallmolecule drugs, and even more particularly oncolytics, which can reducetheir adverse and often toxic side-effects, whilst simultaneouslyimproving their efficacy and ease of formulation. Specifically, there isa need for improved methods for delivering drugs that possess an optimalbalance of bioavailability due to reduced clearance times, bioactivity,and efficacy, coupled with reduced side-effects. The present inventionmeets those needs.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a water-soluble prodrug.The prodrug of the invention comprises a water-soluble polymer havingthree or more arms, typically at least three of which are covalentlyattached to an active agent, e.g., a small molecule. The conjugates ofthe invention provide an optimal balance of polymer size and structurefor achieving improved drug loading, since the conjugates of theinvention generally possess three or more active agents attached,preferably releasably, to a water-soluble polymer. In one embodiment,each arm of the water-soluble polymer possesses an active agentcovalently attached thereto, preferably by a hydrolyzable linkage.

In a particular embodiment, the prodrug conjugate comprises a multi-armpolymer, i.e., having three or more arms, where the conjugate comprisesthe following generalized structure:

R(-Q-POLY₁-X-D)_(q)  I

In structure I, R is an organic radical possessing from about 3 to about150 carbon atoms, preferably from about 3 to about 50 carbon atoms, andeven more preferably from about 3 to about 10 carbon atoms, optionallycontaining one or more heteroatoms (e.g., O, S, or N). In oneembodiment, R possesses a number of carbon atoms selected from the groupconsisting of 3, 4, 5, 6, 7, 8, 9, and 10. R may be linear or cyclic,and typically, emanating therefrom are at least 3 independent polymerarms each typically having at least one active agent moiety covalentlyattached thereto. Looking at the above structure, “q” corresponds to thenumber of polymer arms emanating from “R”.

In structure I, Q is a linker, preferably one that is hydrolyticallystable. Typically, Q contains at least one heteratom such as O, or S, orNH, where the atom proximal to R in Q, when taken together with R,typically represents a residue of the core organic radical R.Illustrative examples are provided herein. Generally, Q contains from 1to about 10 atoms, or from 1 to about 5 atoms. More particularly, Qtypically contains one of the following numbers of atoms: 1, 2, 3, 4, 5,6, 7, 8, 9, or 10. In a particular embodiment, Q is O, S, or —NH—C(O)—.

In structure I, POLY₁ represents a water-soluble and non-peptidicpolymer. Representative polymers include poly(alkylene glycol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharide), poly(α-hydroxy acid), poly(acrylic acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), orcopolymers or terpolymers thereof.

In a particular embodiment of structure I, POLY₁ is a polyethyleneglycol, preferably a linear polyethylene glycol (i.e., in each arm ofthe overall multi-arm structure). In yet another embodiment, POLY₁corresponds to the structure, —(CH₂CH₂O)_(n)—, where n ranges from about10 to about 400, preferably from about 50 to about 350.

In structure I, X is a spacer that comprises a hydrolyzable linkage,where the hydrolyzable linkage is attached directly to the active agent,D. Typically, at least one atom of the hydrolyzable linkage is containedin the active agent, D, in its unmodified form, such that uponhydrolysis of the hydrolyzable linkage comprised within X, the activeagent, D, is released. Generally speaking, the spacer, X, has an atomlength of from about 4 atoms to about 50 atoms, or more preferably fromabout 5 atoms to about 25 atoms, or even more preferably from about 5atoms to about 20 atoms. Representative spacers have a length of fromabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20atoms.

In yet another particular embodiment, X possesses the structure: Y—Z,where Y is a spacer fragment covalently attached to Z, a hydrolyticallydegradable linkage. In certain embodiments, Z itself may not constitutea hydrolytically degradable linkage, however, when taken together withY, or at least a portion of Y, forms a linkage that is hydrolyticallydegradable.

In yet a more particular embodiment of the spacer, X, Y has thestructure: —(CR_(x)R_(y))_(a)—K—(CR_(x)R_(y))_(b)—(CH₂CH₂O)_(c)—,wherein each R_(x) and R_(y), in each occurrence, is independently H oran organic radical selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, and substituted aryl, a ranges from 0 to 12 (i.e., can be0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), b ranges from 0 to 12(i.e., can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), K isselected from —C(O)—, —C(O)NH—, —NH—C(O)—, —O—, —S—, O—C(O)—, C(O)—O—,O—C(O)—O—, O—C—(O)—NH—, NH—C(O)—O—, c ranges from 0 to 25, and Z isselected from C(O)—O—, O—C(O)—O—, —O—C(O)—NH—, and NH—C(O)—O—. Theparticular structure of K and of Z will depend upon the values of eachof a, b, and c, such that none of the following linkages result in theoverall structure of spacer X, —O—O—, NH—O—, NH—NH—.

Preferably, Y comprises (CH₂)_(a)—C(O)NH—(CH₂)_(0,1)—(CH₂CH₂O)₀₋₁₀.

In yet another embodiment of the spacer, X, Y has the structure:—(CR_(x)R_(y))_(a)—K—(CR_(X)R_(y))_(b)—(CH₂CH₂NH)_(c)—, where thevariables have the values previously described. In certain instances,the presence of the short ethylene oxide or ethyl amino fragments inspacer, X, can be useful in achieving good yields during preparation ofthe prodrug conjugate, since the presence of the linker can help tocircumvent problems associated with steric hindrance, due to themulti-armed reactive polymer, the structure of the active agent, or acombination of both. Preferably, c is selected from the group consistingof 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

Preferably, R_(x) and R_(y) in each occurrence are independently H orlower alkyl. In one embodiment, R_(x) and R_(y) are in each occurrenceH. In yet another embodiment, a ranges from 0 to 5. In yet anotherembodiment, b ranges from 0 to 5. In yet another embodiment, c rangesfrom 0 to 10. In yet another embodiment, K is —C(O)—NH. Any of theembodiments described herein is meant to apply not only to generalizedstructure I, but also to extend to particular combinations ofembodiments.

In yet another embodiment, R_(x) and R_(y) in each occurrence are H, ais 1, K is —C(O)—NH, and b is 0 or 1.

Representative examples of X include —CH₂—C(O)—NH—CH₂—C(O)O— (here, Ycorresponds to —CH₂—C(O)—NH—CH₂— and Z corresponds to —C(O)—O—), and—CH₂—C(O)—NH—(CH₂CH₂O)₂—C(O)—O— (here, Y corresponds to—CH₂—C(O)—NH—(CH₂CH₂O)₂— and Z corresponds to —C(O)—O—).

Returning now to structure I, D is an active agent moiety, and q (thenumber of independent polymer arms) ranges from about 3 to about 50.Preferably, q ranges from about 3 to about 25. More preferably, q isfrom 3 to about 10, and possesses a value of 3, 4, 5, 6, 7, 8, 9, or 10.

In accordance with one embodiment of the invention, the conjugatecomprises a polymer having from about 3 to about 25 active agentmolecules covalently attached thereto. More particularly, the conjugatecomprises a water-soluble polymer having 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 active agentmolecules covalently attached thereto. In a further embodiment, theconjugate of the invention has from about 3 to about 8 active agentmolecules covalently attached to the multi-armed water-soluble polymer.Typically, although not necessarily, the number of polymer arms willcorrespond to the number of active agents covalently attached to thewater-soluble polymer.

The active agent moiety, D, is an active agent comprising a functionalgroup suitable for covalent attachment to the multi-armed polymerdescribed herein to form a hydrolyzable linkage, such that uponhydrolysis, the active agent is released in its unmodified form.

Preferred active agent moieties include anticancer agents.

In one embodiment, the active agent is a small molecule. In a particularembodiment, the active agent moiety is a small molecule possessing amolecular weight of less than about 1000. In yet additional embodiments,the small molecule drug possesses a molecular weight of less than about800, or even less than about 750. In yet another embodiment, the smallmolecule drug possesses a molecular weight of less than about 500 or, insome instances, even less than about 300.

In yet another embodiment, the small molecule is an oncolytic drughaving at least one hydroxyl group.

In yet a further embodiment, D represents a camptothecin compound havingthe structure:

wherein R₁-R₅ are each independently selected from the group consistingof hydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxcarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or

R₂ together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy;

R₆ is H or OR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl; and

L is the site of attachment to X.

In yet another particular embodiment, D is irinotecan (structure shownbelow).

In yet another particular embodiment, D is docetaxel.

Alternatively, D is a small molecule selected from the group consistingof platins, oxymorphone analogues, steroids, quinolones, andnucleosides.

In one embodiment, D is a platin such as cis-platin, hydroxyplatin,carboplatin, or oxaliplatin.

In yet a further embodiment, D is an oxymorphone analogue such asnaloxone, methylnaltrexone, oxymorphone, codeine, oxycodone, ormorphone.

In yet an additional embodiment, D is a steroid such as budesonide,triamcinolone, or fluticasone.

In yet another embodiment, D is a quinolone, isoquinolone orfluoroquinolone such as ciprofloxacin, moxifloxacin, or palonosetron.

In yet an additional embodiment, D is a nucleoside or nucleotide such asgemcitabine, cladribine, or fludarabine.

In yet another aspect, the invention encompasses a pharmaceuticalcomposition comprising a multi-armed polymer prodrug as described above.In one embodiment, the composition further comprises a multi-arm polymerprodrug, where one or more of ‘q’ polymer arms is absent D.

More particularly, the invention includes a composition comprising amulti-arm polymer prodrug having the structureR(-Q-POLY₁-X′-D_(0,1))_(q). In the foregoing structure, R, Q, POLY₁, andq are as previously described; X′ is either (i) a spacer, X, comprisinga hydrolyzable linkage, such that upon hydrolysis of said hydrolyzablelinkage, D, is released, or is (ii) X″, a terminal moiety; and D is asmall molecule, where D₁ indicates the presence of D and D₀ indicatesits absence, where the following conditions also apply: if X′ is X, thenD is D₁, and if X′ is X″, then D is D₀. Typically, the compositioncomprises at least one multi-arm polymer prodrug species wherein X′ isX. The terminal moiety, X″, typically corresponds to an unreactedfunctional group present on one or more arms of the multi-armed polymer,or, is an inert derivative thereof, e.g., resulting from work-up, suchas a hydrolysis product.

More particularly, a composition of the invention may comprise one ormore prodrug species having the structure:

R-(Q-POLY₁-X-D₁)_(m)(Q-POLY₁-X″)_(s).

where the variables are as previously described. The preceding structuremay also be represented as follows:

In the above structures (which are identical), m and s are eachintegers, where the sum of m and s is q, and where m and s eachindependently have values ranging from 0 to q. As described above, q(the number of polymer arms emanating from R) typically ranges fromabout 3 to about 50. Preferably, q ranges from about 3 to about 25. Morepreferably, q is from 3 to about 10, and possesses a value of 3, 4, 5,6, 7, 8, 9, or 10.

In one embodiment, the composition possesses an average number of D permulti-arm polymer ranging from 2.1-3.75.

In yet another embodiment, the composition possesses an average numberof D per multi-arm polymer ranging from 2.3-3.5

In another embodiment, the composition comprises a mixture of “q”prodrug species.

In yet a more particular embodiment of a composition in accordance withthe invention, X″ is —CH₂COOH.

Also forming part of the invention is a composition comprising a mixtureof one or more prodrug species having the structure:

wherein O-Irinotecan is a residue of irinotecan as shown below:

m+s=4, and n ranges from 40 to 500.

In yet another embodiment, the invention encompasses a compositioncomprising a mixture of one or more prodrug species having thestructure:

where m+s=4, and n ranges from 40 to 500.

The multi-armed polymer prodrugs of the invention possess many uniquefeatures, particularly in the instance where the small molecule is ananticancer compound. For example, in one embodiment, provided is amulti-arm polymer prodrug, which when evaluated in a suitable animalmodel for solid tumor-type cancers and administered in a therapeuticallyeffective amount, is effective to suppress tumor growth to an extentthat is at least 1.5 times that, or even twice that observed for theunmodified anticancer agent, when evaluated over a time course of 30days. In yet another embodiment, the prodrug is effective to suppresstumor growth to the above extent or even greater when evaluated over atime course of 60 days. The small molecule employed is one known topossess anticancer properties, however, by virtue of its conjugation toa multi-armed polymer as described herein, possesses significantlyimproved efficacy and/or pharmacokinetics in comparison to the smallmolecule, e.g., anticancer compound, itself. Suitable solid tumor typesinclude but are not limited to malignant sarcomas, carcinomas andlymphomas of the breast, ovaries, colon, kidney, bile duct, lung andbrain.

In another aspect, the invention encompasses reactive multi-arm polymerssuitable for preparing any of the above-described prodrug conjugates.

In another aspect, the invention encompasses a pharmaceuticalcomposition comprising a multi-arm polymer prodrug conjugate asdescribed above in combination with a pharmaceutically acceptablecarrier.

Another aspect of the invention provides a method for treating variousmedical conditions in a mammalian subject. More specifically, theinvention encompasses a method of administering to a mammalian subjectin need thereof a therapeutically effective amount of a multi-armprodrug conjugate of the invention. In one embodiment, the drug moiety,D, is an anticancer agent such as a camptothecin (e.g., irinotecan) ordocetaxel, and is effective to suppress tumor growth. In a particularlypreferred embodiment, a multi-armed prodrug conjugate of the invention,particularly one where D is an anticancer agent, exhibits one or more ofthe following characteristics: (i) suppresses tumor growth to an extentgreater than that of unmodified D, (ii) demonstrates a tumor retentiontime that is increased over that of unmodified D, (iii) exhibits a rateof clearance that is reduced in comparison to that of unmodified D,and/or (iv) produces reduced adverse side effects in comparison tounmodified D.

Also provided herein is a method of treating cancer or a viral infection(where D is an anticancer or an antiviral agent, respectively) byadministering a multi-arm polymer conjugate as described herein.

In yet another aspect, the invention provides a method of treating atopoisomerase I inhibitor-related disease in a mammalian subject byadministering a therapeutically effective amount of a multi-arm polymerprodrug to a mammalian subject in need thereof, where the small moleculeis a camptothecin type molecule.

Also forming part of the invention is a method of targeting a solidtumor in a mammalian subject. The method includes the step ofadministering a therapeutically effective amount of a multi-arm polymerprodrug of an anticancer agent known to be effective in the treatment ofsolid tumors to a subject diagnosed as having one or more canceroussolid tumors. As a result of said administering, the prodrug iseffective to produce an inhibition of solid tumor growth in the subjectthat is increased over the inhibition of solid tumor growth resultingfrom administration of the anticancer agent alone.

In a further aspect, a method for preparing a multi-arm polymer prodrugconjugate of the invention is provided. In the method, a small molecule,D, is provided, where the small molecule comprises a functional group,F, suitable for forming a hydrolyzable linkage, Z. The small molecule isreacted with a bifunctional spacer, Y′, comprising each a first and asecond functional group, F1 and F2. The functional group F2 is suitablefor reaction with F, and F1 may optionally be in protected form(F1-Y′-F2). The reaction is carried out under conditions effective toform a partially modified active agent comprising a hydrolyzablelinkage, Z, resulting from reaction of F and F2, which corresponds tothe structure D-Z—Y′—F1. If necessary, the method includes the optionalstep of deprotecting F1 contained in the partially modified activeagent. The method then includes the step of reacting the partiallymodified active agent, D-Z—Y′—F1, with a multi-armed water-solublepolymer comprising the structure, R(-Q-POLY₁-F3)_(q), where R, Q, POLY₁,and Q are as previously defined, and F3 is a functional group that isreactive with F1. The reaction is carried out under conditions effectiveto promote reaction between F3 and F1 to convert Y′ to Y, to therebyform a polymer prodrug having the structure, R(-Q-POLY₁-Y—Z-D)_(q),where Y is a spacer fragment, and Z is a hydrolyzable linkage, which,upon hydrolysis, releases D.

In one embodiment of the method, a stoichiometric excess in an amountgreater than “q” moles of the partially modified active agent,D-Z—Y′—F1, is reacted with the multi-armed water-soluble,R(-Q-POLY₁-F3)_(q) to drive the reaction to completion, i.e., tocovalently attach active agent to each of the reactive polymer arms.

In yet another embodiment, where the small molecule D possessesadditional functional groups reactive with F2, the method furthercomprises the step of protecting the additional functional groups withsuitable protecting groups prior to reaction with the bifunctionalspacer. These protecting groups are then removed from the smallmolecules of the prodrug product, R(-Q-POLY₁-Y—Z-D)_(q).

In yet another embodiment of the above method, the reacting step resultsin formation of additional prodrug species having the structure:

R(-Q-POLY₁-Y—Z-D)_(m)(-Q-POLY₁-X″)_(s)

which can also be represented as

where m and s are integers, each independently ranging in value from 0to q, and the sum of m and s is q, and X″ is a terminal moiety that iseither the same as F3 or is a hydrolysis product or chemical equivalentthereof.

In yet another embodiment, the method additionally comprises the step ofisolating said one or more polymer prodrugs.

According to yet another aspect of the invention, provided is yetanother method for preparing a multi-arm polymer prodrug of theinvention. The method includes the step of providing a reactivemulti-arm polymer having the structure, R(-Q-POLY₁-F3)_(q), where R, Q,POLY₁, and q are as previously described, and F3 is a reactivefunctional group. The multi-arm polymer is then reacted with abifunctional spacer, Y′, comprising each a first and a second functionalgroup, F1 and F2, wherein F1 is suitable for reaction with F3, and F1 isoptionally in protected form (F1-Y′-F2). The reaction is carried outunder conditions effective to form an intermediate multi-arm polymerresulting from reaction of F3 and F1, and having the structure,R(-Q-POLY₁-Y—F2)_(q). The method further includes the optional step ofdeprotecting F2 in the intermediate multi-arm polymer,R(-Q-POLY₁-Y—F2)_(q) if such is in protected form. The intermediatemulti-arm polymer, R(-Q-POLY₁-Y—F2)_(q), is then reacted with a smallmolecule, D, comprising a functional group, F, suitable for forming ahydrolyzable linkage, Z, upon reaction of F with F2, under conditionseffective to thereby form a prodrug having the structure,R(-Q-POLY₁-Y—Z-D)_(q), where Z is a hydrolyzable linkage, which, uponhydrolysis, releases D.

Reactive functional groups such as those described above as F1, F2 andF3, are numerous and may be selected from, for example, hydroxyl, activeester (e.g., N-hydroxysuccinimidyl ester and 1-benzotriazolyl ester),active carbonate (e.g., N-hydroxysuccinimidyl carbonate,1-benzotriazolyl carbonate, p-nitrophenyl carbonate), acid halide,acetal, aldehyde having a carbon length of 1 to 25 carbons (e.g.,acetaldehyde, propionaldehyde, and butyraldehyde), aldehyde hydrate,alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine,hydrazide, thiol, alkanoic acids having a carbon length (including thecarbonyl carbon) of 1 to about 25 carbon atoms (e.g., carboxylic acid,carboxymethyl, propanoic acid, and butanoic acid), isocyanate,isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxal, and dione.

In one embodiment, the bifunctional spacer, Y′ is an amino acid orderived from an amino acid. Representative amino acids have thestructure HO—C(O)—CH(R″)—NH-Gp wherein R″ is H, C1-C6 alkyl, orsubstituted C1-C6 alkyl and Gp is an amino-protecting group. In analternative embodiment, the bifunctional spacer, Y′ possesses thestructure: —C(O)—(OCH₂CH₂)₁₋₁₀—NH-Gp.

In yet another embodiment of the foregoing method, the reacting stepresults in formation of additional prodrug species having the structure,R(-Q-POLY₁-Y—Z-D)_(m)(-Q-POLY₁-X″)_(s), where m and s are integers eachindependently having a value ranging from 0 to q, wherein the sum of mand s is q, and X″ is a terminal moiety that is either the same as F2 oris a hydrolysis product or chemical equivalent thereof.

The above methods for preparing a prodrug of the invention may includethe additional steps of purifying the intermediates and/or the finalprodrug products, for example by size exclusion chromatography or ionexchange chromatography in instances in which the compounds to bepurified contain one or more ionizable groups, such as carboxyl oramino.

Each of the herein-described features of the invention is meant to applyequally to each and every embodiment as described herein, unlessotherwise indicated.

These and other objects and features of the invention will become morefully apparent when read in conjunction with the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the effect of an exemplary multi-armPEG-irinotecan conjugate, 4-arm-PEG-GLY-IRINO-20k, on the growth of HT29human colon tumors implanted in athymic nude mice in comparison to anuntreated control group and a group treated with irinotecan as describedin detail in Example 2.

FIG. 2 is a graph illustrating the effects of a variety of doses (90mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary multi-arm PEG irinotecanconjugate, 4-arm-PEG-GLY-IRINO-20k on the growth of NCI-H460 human lungtumors implanted in athymic nude mice in comparison to a control groupand a group treated with irinotecan as described in Example 6.

FIG. 3 is a graph illustrating the effects of a variety of doses (90mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 40 kilodalton (40K)multi-arm PEG irinotecan conjugate, 4-arm-PEG-GLY-IRINO-40k, on thegrowth of NCI-H460 human lung tumors implanted in athymic nude mice incomparison to a control group and a group treated with irinotecan asdescribed in Example 6.

FIG. 4 is a graph illustrating the effects of a variety of doses (90mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 20 kilodalton (20K)multi-arm PEG-irinotecan conjugate, 4-arm-PEG-GLY-IRINO-20k, on thegrowth of HT29 human colon tumors implanted in athymic nude mice incomparison to an untreated control group and a group treated withirinotecan as described in detail in Example 6.

FIG. 5 is a graph illustrating the effects of a variety of doses (90mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 40 kilodalton (40K)multi-arm PEG-irinotecan conjugate, 4-arm-PEG-GLY-IRINO-40k, on thegrowth of HT29 human colon tumors implanted in athymic nude mice incomparison to an untreated control group and a group treated withirinotecan as described in detail in Example 6.

FIG. 6 is a graph illustrating the concentration in venous plasma overtime of (i) an exemplary 20 kilodalton (20K) multi-arm PEG irinotecanconjugate, 4-arm-PEG-GLY-IRINO-20k, and (ii) a 40 kilodalton multi-armPEG irinotecan conjugate, 4-arm-PEG-GLY-IRINO-40k, following IVadministration as a single dose in athymic nude mice implanted witheither HT29 human colon tumors or NCI-H460 human lung tumors asdescribed in Example 7.

FIG. 7 is a graph illustrating the concentration in tumor tissue overtime of (i) an exemplary 20 kilodalton (20K) multi-arm PEG irinotecanconjugate, 4-arm-PEG-GLY-IRINO-20k, and (ii) a 40 kilodalton multi-armPEG irinotecan conjugate, 4-arm-PEG-GLY-IRINO-40k, following IVadministration as a single dose in athymic nude mice implanted with HT29human colon tumors as described in Example 7.

FIG. 8 is a graph illustrating the concentration of4-arm-PEG-GLY-SN-38-20k or 4-arm-PEG-GLY-SN-38-40k in plasma over timefollowing IV administration of (i) an exemplary 20 kilodalton (20K)multi-arm PEG irinotecan conjugate, or (ii) a 40 kilodalton multi-armPEG irinotecan conjugate, respectively, as a single dose in athymic nudemice implanted with HT29 human colon tumors as described in Example 7.

FIG. 9 is a graph illustrating the concentration of4-arm-PEG-GLY-SN-38-20k or 4-arm-PEG-GLY-SN-38-40k in tumor tissue overtime following IV administration of (i) an exemplary 20 kilodalton (20K)multi-arm PEG irinotecan conjugate, or (ii) a 40 kilodalton multi-armPEG irinotecan conjugate, respectively, as a single dose in athymic nudemice implanted with HT29 human colon tumors as described in Example 7.

FIG. 10 is a graph illustrating the concentration of irinotecan invenous plasma over time following IV administration of (i) an exemplary20 kilodalton (20K) multi-arm PEG irinotecan conjugate,4-arm-PEG-GLY-IRINO-20k, or (ii) a 40 kilodalton multi-arm PEGirinotecan conjugate, 4-arm-PEG-GLY-IRINO-40k, or (iii) irinotecanitself as a single dose in athymic nude mice implanted with HT29 humancolon tumors as described in Example 7.

FIG. 11 is a graph illustrating the concentration of irinotecan in tumortissue over time following IV administration of (i) an exemplary 20kilodalton (20K) multi-arm PEG irinotecan conjugate,4-arm-PEG-GLY-IRINO-20k, or (ii) a 40 kilodalton multi-arm PEGirinotecan conjugate, 4-arm-PEG-GLY-IRINO-40k, or (iii) irinotecanitself, as a single dose in athymic nude mice implanted with HT29 humancolon tumors as described in Example 7.

FIG. 12 is a graph illustrating the concentration of SN-38 in plasmaover time following IV administration of (i) an exemplary 20 kilodalton(20K) multi-arm PEG irinotecan conjugate, 4-arm-PEG-GLY-IRINO-20k, or(ii) a 40 kilodalton multi-arm PEG irinotecan conjugate,4-arm-PEG-GLY-IRINO-40k, or (iii) irinotecan itself, as a single dose inathymic nude mice implanted with HT29 human colon tumors as described inExample 7.

FIG. 13 is a graph illustrating the concentration of SN-38 in tumortissue over time following IV administration of (i) an exemplary 20kilodalton (20K) multi-arm PEG irinotecan conjugate,4-arm-PEG-GLY-IRINO-20k, or (ii) a 40 kilodalton multi-arm PEGirinotecan conjugate, 4-arm-PEG-GLY-IRINO-40k, or (iii) irinotecanitself, as a single dose in athymic nude mice implanted with HT29 humancolon tumors as described in Example 7.

FIG. 14 is a graph illustrating the in vitro release of irinotecan from4-arm-PEG-GLY-IRINO-20K in phosphate buffer (pH 7.4, 37° C.) over timeas described in detail in Example 10.

FIGS. 15A and 15B are plots demonstrating the mean tumor volume (i.e.,tumor growth) over time in athymic mice implanted with MCF-7 humanbreast tumors following administration of 4-arm-PEG-GLY-IRINO-20K (FIG.15A) or irinotecan (FIG. 15B) as described in Example 11.

FIG. 15C contains plots comparing mean tumor weight (mg) over time(days) in athymic mice implanted with MCF-7 human breast tumorsfollowing administration of 4-arm-PEG-GLY-IRINO-20K, irinotecan, or acontrol. Subjects were administered 20 mg/kg irinotecan equivalent perdose; dosing occurred every four days for a total of three dosesadministered.

FIGS. 16A, 16B and 16C are plots illustrating the results of a doseranging study to evaluate the effect of a single dose of docetaxelversus 4-arm-PEG-GLY-DOC-20K on H-460 non-small cell lung cancer tumorsuppression in female xenograft athymic nude mice over time. Dosageamounts were as follows: 10 mg/kg (based upon docetaxel dosage) (FIG.16A), 20 mg/kg (FIG. 16B), and 40 mg/kg (FIG. 16C). Details of the studyare provided in Example 16.

FIG. 17 is a plot demonstrating the effects of two doses of each ofthree different dosage amounts of docetaxel (10 mg/kg, 20 mg/kg, and 30mg/kg) and 4-arm-PEG-GLY-DOC-20K (20 mg/kg, 40 mg/kg, and 60 mg/kg) infemale xenograft athymic nude mice implanted with H460 non-small celllung cancer tumors over time as described in Example 16.

FIG. 18 is a plot demonstrating the effects of two doses of each ofthree different dosage amounts of docetaxel (10 mg/kg, 20 mg/kg, and 30mg/kg) and 4-arm-PEG-GLY-DOC-20K (20 mg/kg, 40 mg/kg, and 60 mg/kg) infemale xenograft athymic nude mice implanted with DU-145 prostate tumorsover time as described in Example 17.

FIG. 19 is a plot demonstrating the anti-tumor effect of docetaxel and4-arm-PEG-GLY-DOC-20K, respectively, over time in mice implanted withMCF-7 breast tumors as described in Example 18.

FIG. 20 illustrates exposure to SN-38 in HT-29 colorectal tumors in miceresulting from administration of 4-arm-PEG-gly-irino-20K.

FIG. 21 is a plot demonstrating reduced neutropenia in dogs administered4-arm-PEG-GLY-IRINO-20K when compared to dogs administered irinotecan atan equivalent dose.

FIG. 22 is a bar graph illustrating the maximum tolerated dose (MTR) of4-arm-PEG-GLY-IRINO-20K when compared to irinotecan in both rat and dog.

FIG. 23 is a bar graph illustrating an additional advantage ofadministering 4-arm-PEG-GLY-IRINO-20K when compared to irinotecan: fewerdiarrhea episodes in dogs.

FIG. 24 is a plot illustrating plasma levels of SN-38 in dogsadministered 4-arm-PEG-GLY-IRINO-20K or irinotecan. SN-38 levels in dogsadministered 4-arm-PEG-GLY-IRINO-20K are elevated and sustained over alonger period of time when compared to SN-38 levels in dogs administeredunmodified irinotecan.

FIG. 25 provides a pharmacokinetic profile resulting from administrationof a single dose of 4-arm-PEG-GLY-IRINO-20K to a breast cancer patientin comparison to the profile resulting from administration ofirinotecan.

FIG. 26 is a plot demonstrating the neutrophil effects of4-arm-PEG-GLY-DOC-20K versus docetaxel when administered to dogs, where4-arm-PEG-GLY-DOC-20K resulted in less neutropenia than docetaxel whenadministered at a higher single dose.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art.

DEFINITIONS

It must be noted that, as used in this specification, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to a “polymer” includesa single polymer as well as two or more of the same or differentpolymers, reference to a “conjugate” refers to a single conjugate aswell as two or more of the same or different conjugates, reference to an“excipient” includes a single excipient as well as two or more of thesame or different excipients, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

A “functional group” is a group that may be used, under normalconditions of organic synthesis, to form a covalent linkage between thestructure to which it is attached and another structure, which typicallybears a further functional group. The functional group generallyincludes multiple bond(s) and/or heteroatom(s). Preferred functionalgroups for use in the polymers of the invention are described below.

The term “reactive” refers to a functional group that reacts readily orat a practical rate under conventional conditions of organic synthesis.This is in contrast to those groups that either do not react or requirestrong catalysts or impractical reaction conditions in order to react(i.e., a “nonreactive” or “inert” group).

“Not readily reactive”, with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions effective to produce a desired reactionin the reaction mixture.

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative which reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters include, forexample, imidazolyl esters, and benzotriazole esters, and imide esters,such as N-hydroxysuccinimidyl (NHS) esters. An activated derivative maybe formed in situ by reaction of a carboxylic acid with one of variousreagents, e.g. benzotriazol-1-yloxy tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), preferably used in combination with1-hydroxy benzotriazole (HOBT) or 1-hydroxy-7-azabenzotriazole (HOAT);O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU); or bis(2-oxo-3-oxazolidinyl)phosphinicchloride (BOP-Cl).

A “chemical equivalent” of a functional group is one that possessesessentially the same type of reactivity as the functional group. Forinstance, one functional group that undergoes an SN2 reaction isconsidered to be a functional equivalent of another such functionalgroup.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups thatmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof is meant to encompass protected forms thereof.

“PEG” or “poly(ethylene glycol)” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Typically, PEGs for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. The variable (n) is 3 to 3000, and theterminal groups and architecture of the overall PEG may vary. When PEGfurther comprises a spacer as in structure I above (to be described ingreater detail below), the atoms comprising the spacer (X), whencovalently attached to a PEG segment, do not result in formation of (i)an oxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) anitrogen-oxygen bond (N—O, O—N). “PEG” means a polymer that contains amajority, that is to say, greater than 50%, of subunits that are—CH₂CH₂O—. PEGs for use in the invention include PEGs having a varietyof molecular weights, structures or geometries to be described ingreater detail below.

“Water-soluble”, in the context of a polymer of the invention or a“water-soluble polymer segment” is any segment or polymer that issoluble in water at room temperature. Typically, a water-soluble polymeror segment will transmit at least about 75%, more preferably at leastabout 95% of light, transmitted by the same solution after filtering. Ona weight basis, a water-soluble polymer or segment thereof willpreferably be at least about 35% (by weight) soluble in water, morepreferably at least about 50% (by weight) soluble in water, still morepreferably about 70% (by weight) soluble in water, and still morepreferably about 85% (by weight) soluble in water. It is most preferred,however, that the water-soluble polymer or segment is about 95% (byweight) soluble in water or completely soluble in water.

An “end-capping” or “end-capped” group is an inert group present on aterminus of a polymer such as PEG. An end-capping group is one that doesnot readily undergo chemical transformation under typical syntheticreaction conditions. An end capping group is generally an alkoxy group,—OR, where R is an organic radical comprised of 1-20 carbons and ispreferably lower alkyl (e.g., methyl, ethyl) or benzyl. “R” may besaturated or unsaturated, and includes aryl, heteroaryl, cyclo,heterocyclo, and substituted forms of any of the foregoing. Forinstance, an end capped PEG will typically comprise the structure“RO—(CH₂CH₂O)_(n)—”, where R is as defined above. Alternatively, theend-capping group can also advantageously comprise a detectable label.When the polymer has an end-capping group comprising a detectable label,the amount or location of the polymer and/or the moiety (e.g., activeagent) to which the polymer is coupled, can be determined by using asuitable detector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like.

“Non-naturally occurring” with respect to a polymer of the inventionmeans a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer of the invention may however contain oneor more subunits or segments of subunits that are naturally occurring,so long as the overall polymer structure is not found in nature.

“Molecular mass” in the context of a water-soluble polymer of theinvention such as PEG, refers to the nominal average molecular mass of apolymer, typically determined by size exclusion chromatography, lightscattering techniques, or intrinsic velocity determination in1,2,4-trichlorobenzene. Molecular weight in the context of awater-soluble polymer, such as PEG, can be expressed as either anumber-average molecular weight or a weight-average molecular weight.Unless otherwise indicated, all references to molecular weight hereinrefer to the weight-average molecular weight. Both molecular weightdeterminations, number-average and weight-average, can be measured usinggel permeation chromatographic or other liquid chromatographictechniques. Other methods for measuring molecular weight values can alsobe used, such as the use of end-group analysis or the measurement ofcolligative properties (e.g., freezing-point depression, boiling-pointelevation, or osmotic pressure) to determine number-average molecularweight or the use of light scattering techniques, ultracentrifugation orviscometry to determine weight-average molecular weight. The polymers ofthe invention are typically polydisperse (i.e., number-average molecularweight and weight-average molecular weight of the polymers are notequal), possessing low polydispersity values such as less than about1.2, less than about 1.15, less than about 1.10, less than about 1.05,and less than about 1.03. As used herein, references will at times bemade to a single water-soluble polymer having either a weight-averagemolecular weight or number-average molecular weight; such referenceswill be understood to mean that the single-water soluble polymer wasobtained from a composition of water-soluble polymers having the statedmolecular weight.

The term “linker” is used herein to refer to an atom or a collection ofatoms used to link interconnecting moieties, such as an organic radicalcore and a polymer segment, POLY₁. A linker moiety may be hydrolyticallystable or may include a physiologically hydrolyzable or enzymaticallydegradable linkage. A linker designated herein as Q is hydrolyticallystable.

The term “spacer” is used herein to refer to a collection of atoms usedto link interconnecting moieties, such as POLY₁ and the active agent, D.A spacer moiety may be hydrolytically stable or may include aphysiologically hydrolyzable or enzymatically degradable linkage. Aspacer designated herein as X comprises a hydrolyzable linkage, wherethe hydrolyzable linkage is attached directly to the active agent, D,such that upon hydrolysis, the active agent is released in its parentform.

A “hydrolyzable” bond is a relatively weak bond that reacts with water(i.e., is hydrolyzed) under physiological conditions. The tendency of abond to hydrolyze in water will depend not only on the general type oflinkage connecting two central atoms but also on the substituentsattached to these central atoms. Illustrative hydrolytically unstablelinkages include carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides andoligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes. Such a linkage requires the actionof one or more enzymes to effect degradation.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Multi-armed” in reference to the geometry or overall structure of apolymer refers to polymer having 3 or more polymer-containing “arms”.Thus, a multi-armed polymer may possess 3 polymer arms, 4 polymer arms,5 polymer arms, 6 polymer arms, 7 polymer arms, 8 polymer arms or more,depending upon its configuration and core structure. One particular typeof highly branched polymer is a dendritic polymer or dendrimer, that,for the purposes of the invention, is considered to possess a structuredistinct from that of a multi-armed polymer.

“Branch point” refers to a bifurcation point comprising one or moreatoms at which a polymer splits or branches from a linear structure intoone or more additional polymer arms. A multi-arm polymer may have onebranch point or multiple branch points.

A “dendrimer” is a globular, size monodisperse polymer in which allbonds emerge radially from a central focal point or core with a regularbranching pattern and with repeat units that each contribute a branchpoint. Dendrimers exhibit certain dendritic state properties such ascore encapsulation, making them unique from other types of polymers.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater of some given quantity.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl when three or more carbon atoms are referenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. For substitutions on a phenyl ring,the substituents may be in any orientation (i.e., ortho, meta, or para).

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),preferably C₁-C₇.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 1 to 15 atoms in length, containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl,octynyl, decynyl, and so forth.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents.

“Electrophile” refers to an ion, atom, or collection of atoms that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms that maybe ionic, having a nucleophilic center, i.e., a center that is seekingan electrophilic center, and capable of reacting with an electrophile.

“Active agent” as used herein includes any agent, drug, compound, andthe like which provides some pharmacologic, often beneficial, effectthat can be demonstrated in-vivo or in vitro. As used herein, theseterms further include any physiologically or pharmacologically activesubstance that produces a localized or systemic effect in a patient.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a PEG-active agent conjugate present in apharmaceutical preparation that is needed to provide a desired level ofactive agent and/or conjugate in the bloodstream or in a target tissue.The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofpharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

“Multi-functional” in the context of a polymer of the invention means apolymer having 3 or more functional groups, where the functional groupsmay be the same or different, and are typically present on the polymertermini. Multi-functional polymers of the invention will typicallycontain from about 3-100 functional groups, or from 3-50 functionalgroups, or from 3-25 functional groups, or from 3-15 functional groups,or from 3 to 10 functional groups, i.e., contains 3, 4, 5, 6, 7, 8, 9 or10 functional groups. Typically, in reference to a polymer precursorused to prepare a polymer prodrug of the invention, the polymerpossesses 3 or more polymer arms having at the terminus of each arm afunctional group suitable for coupling to an active agent moiety via ahydrolyzable linkage.

“Difunctional” or “bifunctional” as used interchangeable herein means anentity such as a polymer having two functional groups contained therein,typically at the polymer termini. When the functional groups are thesame, the entity is said to be homodifunctional or homobifunctional.When the functional groups are different, the polymer is said to beheterodifunctional or heterobifunctional.

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

“Polyolefinic alcohol” refers to a polymer comprising an olefin polymerbackbone, such as polyethylene, having multiple pendant hydroxyl groupsattached to the polymer backbone. An exemplary polyolefinic alcohol ispolyvinyl alcohol.

As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer may includea minor number of peptide linkages spaced along the repeat monomersubunits, such as, for example, no more than about 1 peptide linkage perabout 50 monomer units.

The terms “subject”, “individual” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murines, rodents, simians, humans, farm animals,sport animals and pets. Such subjects are typically suffering from orprone to a condition that can be prevented or treated by administrationof a polymer of the invention, typically but not necessarily in the formof a polymer-active agent conjugate as described herein.

The term “about”, particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

“Treatment” or “treating” of a particular condition includes: (1)preventing such a condition, i.e. causing the condition not to develop,or to occur with less intensity or to a lesser degree in a subject thatmay be exposed to or predisposed to the condition but does not yetexperience or display the condition, (2) inhibiting the condition, i.e.,arresting the development or reversing the condition.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

A “small molecule” may be defined broadly as an organic, inorganic, ororganometallic compound typically having a molecular weight of less thanabout 1000. Small molecules of the invention encompass oligopeptides andother biomolecules having a molecular weight of less than about 1000.

An “active agent moiety” in reference to a prodrug conjugate of theinvention, refers to the portion or residue of the unmodified parentactive agent up to the covalent linkage resulting from covalentattachment of the drug (or an activated or chemically modified formthereof) to a polymer of the invention. Upon hydrolysis of thehydrolyzable linkage between the active agent moiety and the multi-armedpolymer, the active agent per se is released.

Multi-Arm Polymer Prodrug Conjugates—Overview

As described generally above, the polymer conjugates of the inventioncomprise a multi-arm water-soluble and non-peptidic polymer covalentlyattached to at least three active agent compounds. The conjugates of theinvention are typically prodrugs, meaning that the active agent,attached to the polymer via a hydrolytically degradable linkage, isreleased over time following administration of the conjugate to asubject. Moreover, the conjugates of the invention arewell-characterized, isolable, and purifiable compositions, in comparisonto, for example, a degradable polymer-matrix having molecules of drugencapsulated therein. The conjugates of the invention exhibit higherdrug loading characteristics when compared to their linear polymer-basedcounterparts, thus lowering the total dosage weight needed to treat aparticular disease state. That is to say, the polymer scaffold of theinvention is effective to covalently attach multiple active agentmolecules thereto, thereby allowing a greater amount of therapeuticagent (i.e., active agent) to be administered per given weight ofpolymer when compared to a linear monofunctional or bifunctional polymerof about the same size but having only one or two active agent moleculesattached thereto. The polymers employed in the invention are hydrophilicin nature, thereby imparting hydrophilicity to the resulting conjugates,which, particularly in the case of water-insoluble active agents,facilitates their formulation into useful pharmaceutical compositions.

Typically, the total number average molecular weight of the overallmulti-arm polymer portion of a polymer conjugate of the invention isabout 800 daltons (Da) to about 100,000 Da, more preferably about 10,000Da to about 60,000 Da, most preferably about 15,000 to about 60,000 Da.Multi-armed polymers having a number average molecular weight of about5,000 Da, about 8,000 Da, about 10,000 Da, about 12,000 Da, about 15,000Da, about 20,000 Da, about 25,000 Da, about 30,000 Da, about 35,000 Da,about 40,000 Da, about 45,000 Da, about 50,000 Da, and about 60,000 Da,among others, are particularly preferred. Multi-armed polymers having amolecular weight of 20,000 Da or greater, i.e., of about 20,000 Da, or25,000 Da, or 30,000 Da, or 40,000 Da or 50,000 Da, or 60,000 Da, areparticularly preferred for tumor-targeting applications. The actualmolecular weight of the multi-armed polymer will depend, of course, onthe number of polymer arms and the molecular weight of each polymer armin the overall multi-armed polymer, as well as the degree ofpolydispersity of the polymer.

The linkage between the multi-armed polymer portion and the active agentis preferably hydrolytically degradable for in vivo release of theparent drug molecule over time. Representative hydrolytically degradablelinkages corresponding to X in structure I include carboxylate ester,carbonate ester, phosphate ester, anhydride, acetal, ketal, acyloxyalkylether, imine, orthoester, and oligonucleotides. Esters such ascarboxylate and carbonate esters are particularly preferred linkages.The particular linkage and linkage chemistry employed will depend uponthe particular active agent, the presence of additional functionalgroups within the active agent, and the like, and can be readilydetermined by one skilled in the art based upon the guidance presentedherein.

With respect to the multi-arm prodrug conjugates of the invention, it isnot necessary for the polymer conjugate itself to exhibit biologicalactivity, since the parent drug is released upon hydrolysis. However, incertain embodiments, the polymer conjugate maintains at least ameasurable degree of activity. That is to say, in some instances, amulti-armed polymer conjugate possesses anywhere from about 1% to about100% or more of the specific activity of the unmodified parent compound.That is to say, a multi-armed polymer prodrug of the invention willpossess from about 1% to about 100% bioactivity relative to theunmodified parent active agent, prior to conjugation. Such activity maybe determined using a suitable in-vivo or in-vitro model, depending uponthe known activity of the particular parent compound. For anticancerdrugs, in vivo anticancer activity is typically evaluated by comparisonof growth rates of tumor implants in drug treated and control groups ofathymic mice using well-established animal models (See for example,Examples 2, 6, 7, 11, 12, 13, 16, 17, and 18). Anticancer activity isindicated by slower tumor growth rates in the treated group relative tothe control group (J. W. Singer, et al., Ann. N.Y. Acad. Sci., 922:136-150, 2000). In general, certain polymer conjugates of the inventionwill possess a specific activity of at least about 2%, 5%, 10%, 15%,25%, 30%, 40%, 50%, 60%, 80%, 90% or more relative to that of theunmodified parent drug when measured in a suitable model.

As demonstrated in Examples 2, 6, 7, 11, 12, 13, 16, 17 and 18,preferred polymer prodrug conjugates of the invention exhibit enhancedproperties in comparison to their unmodified parent drug counterparts.The polymer conjugates of the invention exhibit enhanced permeation andretention (EPR) in target tissues by passively accumulating in suchtissues, to provide targeted delivery of the drug to desired sites inthe body (See Matsumara Y, Maeda H. “A NEW CONCEPT FOR MACROMOLECULARTHERAPEUTICS IN CANCER THERAPY; MECHANISM OF TUMORITROPIC ACCUMULATIONOF PROTEINS AND THE ANTITUMOUR AGENT SMANCS”, Cancer Res 1986;46:6387-92). Moreover, the growth rate of several different types ofcancerous tumors when examined in various in-vivo models wassignificantly decreased following administration of illustrativeconjugates of the invention when compared to unmodified drug.

Additionally, the severity of the side effects associated withadministration of the polymer conjugates of the invention is preferablycomparable to, or even more preferably, is less than, the side effectsassociated with administration of the parent compound. In particular,preferred conjugates and conjugate compositions, particularly thosecomprising about 3 or more molecules of an anticancer agent such asirinotecan per polymer core, when administered to a patient, result inreduced or ameliorated side effects, which may be one or more ofleucopenia, neutropenia, and diarrhea, when compared to the unmodifiedparent drug molecule. The severity of side effects of anticancer agentssuch as camptothecin and camptothecin-like compounds can be readilyassessed (See, for example, Kado, et al., Cancer Chemotherapy andPharmacology, Aug. 6, 2003). The polymer conjugates of the invention arebelieved to exhibit reduced side effects as compared to the unconjugatedparent drug, in part, due to the accumulation of the conjugate moleculesin the target tissue and away from other sites of likely toxicity. Eachof these features of the prodrugs of the invention will now be discussedin greater detail below.

Structural Features of the Polymer Prodrug

As described above, a prodrug of the invention comprises a multi-armpolymer, i.e., having three or more arms, where the conjugate comprisesthe following generalized structure:

R(-Q-POLY₁-X-D)_(q)  I

Each arm of the multi-armed prodrug is independent from the other. Thatis to say, each of the “q” arms of the prodrug may be composed of adifferent Q, POLY₁, X, D and so forth. Typical of such embodiments, ageneralized structure corresponds to:R[(-Q₁-POLY_(1A)-X₁-D₁)(Q₂-POLY_(1B)-X₂-D₂)(Q₃-POLY_(1c)-X₃-D₃)] and soforth for each of the arms emanating from the central organic core.Generally, however, each arm of the multi-armed prodrug is the same.

Each of the variable components of structure I will now be described indetail.

Organic Core, “R”

In structure I, R is an organic core radical possessing from about 3 toabout 150 carbon atoms. Preferably, R contains from about 3 to about 50carbon atoms, and even more preferably, R contains from about 3 to about10 carbon atoms. That is to say, R may possess a number of carbon atomsselected from the group consisting of 3, 4, 5, 6, 7, 8, 9, and 10. Theorganic core may optionally contain one or more heteroatoms (e.g., O, S,or N), depending of course on the particular core molecule employed. Rmay be linear or cyclic, and typically, emanating therefrom are at least3 independent polymer arms, three or more of which have at least oneactive agent moiety covalently attached thereto. Looking at Structure I,“q” corresponds to the number of polymer arms emanating from “R”. Insome instances one or more of the polymer arms may not have an activeagent covalently attached thereto, but rather may have a relativelyunreactive or unreacted functional group at its terminus, typicallyresulting from a synthesis that has failed to go to completion. In thisinstance, D is absent and the individual structure of at least one ofthe polymer arms is in its precursor form (or is a derivative thereof),i.e., having at its terminus not an active agent, D, but rather, afunctional group.

The central core organic radical, R, is derived from a molecule thatprovides a number of polymer attachment sites approximately equal to thedesired number of water-soluble and non-peptidic polymer arms.Preferably, the central core molecule of the multi-arm polymer structureis the residue of a polyol, polythiol, or a polyamine bearing at leastthree hydroxyl, thiol, or amino groups available for polymer attachment.A “polyol” is a molecule comprising a plurality (greater than 2) ofavailable hydroxyl groups. A “polythiol” is a molecule that possesses aplurality (greater than 2) thiol groups. A “polyamine” is a moleculecomprising a plurality (greater than 2) of available amino groups.Depending on the desired number of polymer arms, the precursor polyol,polyamine or polythiol, (prior to covalent attachment of POLY₁) willtypically contain 3 to about 25 hydroxyl, or amino groups or orthiolgroups, respectively, preferably from 3 to about 10 hydroxyl, aminogroups or thiol groups, (i.e., 3, 4, 5, 6, 7, 8, 9, 10), mostpreferably, will contain from 3 to about 8 (e.g., 3, 4, 5, 6, 7, or 8)hydroxyl, amino groups or thiol groups suitable for covalent attachmentof POLY₁. The polyol, polyamine or polythiol may also include otherprotected or unprotected functional groups. Focusing on organic coresderived from polyols or polyamines, although the number of interveningatoms between each hydroxyl or amino group will vary, preferred coresare those having a length of from about 1 to about 20 intervening coreatoms, such as carbon atoms, between each hydroxyl or amino group,preferably from about 1 to about 5. In referring to intervening coreatoms and lengths, —CH₂—, for example, is considered as having a lengthof one intervening atom, although the methylene group itself containsthree atoms total, since the Hs are substituents on the carbon, and—CH₂CH₂—, for instance, is considered as having a length of two carbonatoms, etc. The particular polyol or polyamine precursor depends on thedesired number of polymer arms in the final conjugate. For example, apolyol or polyamine core molecule having 4 functional groups, Q, issuitable for preparing a prodrug in accordance with structure I havingfour polymer arms extending therefrom and covalently attached to activeagent.

The precursor polyol or polyamine core will typically possess astructure R—(OH)_(p) or R—(NH₂)_(p) prior to functionalization with apolymer. The value of p corresponds to the value of q in structure I,since each functional group, typically —OH or —NH₂, in the parent coreorganic molecule, if sterically accessible and reactive, is covalentlyattached to a polymer arm, POLY₁. Note that in structure I, the variable“Q”, when taken together with R, typically represents a residue of thecore organic radical as described herein. That is to say, whendescribing preferred organic core molecules, particularly by name, thecore molecules are described in their precursor form, rather than intheir radical form after removal of, for example, a proton. So, if forexample, the organic core radical is derived from pentaerythritol, theprecursor polyol possesses the structure C(CH₂OH)₄, and the organic coreradical, together with Q, corresponds to C(CH₂O—)₄, where Q is O.

Illustrative polyols that are preferred for use as the polymer coreinclude aliphatic polyols having from 1 to 10 carbon atoms and from 1 to10 hydroxyl groups, including for example, ethylene glycol, alkanediols, alkyl glycols, alkylidene alkyl diols, alkyl cycloalkane diols,1,5-decalindiol, 4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidenediols, dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphaticpolyols include straight chained or closed-ring sugars and sugaralcohols, such as mannitol, sorbitol, inositol, xylitol, quebrachitol,threitol, arabitol, erythritol, adonitol, dulcitol, facose, ribose,arabinose, xylose, lyxose, rhamnose, galactose, glucose, fructose,sorbose, mannose, pyranose, altrose, talose, tagitose, pyranosides,sucrose, lactose, maltose, and the like. Additional examples ofaliphatic polyols include derivatives of glyceraldehyde, glucose,ribose, mannose, galactose, and related stereoisomers. Aromatic polyolsmay also be used, such as 1,1,1-tris(4′-hydroxyphenyl) alkanes, such as1,1,1-tris(4-hydroxyphenyl)ethane, (1,3-adamantanediyl)diphenol,2,6-bis(hydroxyalkyl)cresols,2,2′alkylene-bis(6-t-butyl-4-alkylphenols),2,2′-alkylene-bis(t-butylphenols), catechol, alkylcatechols, pyrogallol,fluoroglycinol, 1,2,4-benzenetriol, resorcinol, alkylresorcinols,dialkylresorcinols, orcinol monohydrate, olivetol, hydroquinone,alkylhydroquinones, 1,1-bi-2-naphthol, phenyl hydroquinones,dihydroxynaphthalenes, 4,4′-(9-fluorenylidene)-diphenol, anthrarobin,dithranol, bis(hydroxyphenyl)methane biphenols, dialkylstilbesterols,bis(hydroxyphenyl)alkanes, bisphenol-A and derivatives thereof,meso-hexesterol, nordihydroguaiaretic acid, calixarenes and derivativesthereof, tannic acid, and the like. Other core polyols that may be usedinclude crown ethers, cyclodextrins, dextrins and other carbohydrates(e.g., monosaccharides, oligosaccharides, and polysaccharides, starchesand amylase).

Preferred polyols include glycerol, trimethylolpropane, reducing sugarssuch as sorbitol or pentaerythritol, and glycerol oligomers, such ashexaglycerol. A 21-arm polymer can be synthesized usinghydroxypropyl-β-cyclodextrin, which has 21 available hydroxyl groups.

Exemplary polyamines include aliphatic polyamines such as diethylenetriamine, N,N′,N″-trimethyldiethylene triamine, pentamethyl diethylenetriamine, triethylene tetramine, tetraethylene pentamine, pentaethylenehexamine, dipropylene triamine, tripropylene tetramine,bis-(3-aminopropyl)-amine, bis-(3-aminopropyl)-methylamine, andN,N-dimethyl-dipropylene-triamine. Naturally occurring polyamines thatcan be used in the present invention include putrescine, spermidine, andspermine. Numerous suitable pentamines, tetramines, oligoamines, andpentamidine analogs suitable for use in the present invention aredescribed in Bacchi et al., Antimicrobial Agents and Chemotherapy,January 2002, p. 55-61, Vol. 46, No. 1, which is incorporated byreference herein.

Provided below are illustrative structures corresponding to the organicradical portion of the conjugate, R, and the corresponding idealizedconjugate, assuming that each of the hydroxyls in the parent polyol hasbeen transformed to a polymer arm and that each polymer arm has drugcovalently attached thereto. Note that the organic radicals shown below,derived from polyols, include the oxygens, which, in the context ofstructure I, for the arms that are polymer arms, are considered as partof Q. It is not necessary that all hydroxyls in, for example, apolyol-derived organic radical, form part of a polymer arm. In theillustrative examples below, Q is shown as 0, but can equally beconsidered as corresponding to S, —NH—, or —NH—C(O)—.

Organic Radical* Illustrative Conjugate

see conjugate below

m = 0-40, preferably 0-10, or 0-5. *includes Q Linkages, Q and X.

The linkages between the organic radical, R, and the polymer segment,POLY₁, or between POLY₁ and the active agent, D, result from thereaction of various reactive groups contained within R, POLY₁, and D.The particular coupling chemistry employed will depend upon thestructure of the active agent, the potential presence of multiplefunctional groups within the active molecule, the need forprotection/deprotection steps, the chemical stability of the activeagent, and the like, and will be readily determined by one skilled inthe art based upon the guidance herein. Illustrative linking chemistryuseful for preparing the polymer conjugates of the invention can befound, for example, in Wong, S. H., (1991), “Chemistry of ProteinConjugation and Crosslinking”, CRC Press, Boca Raton, Fla. and inBrinkley, M. (1992) “A Brief Survey of Methods for Preparing ProteinConjugates with Dyes, Haptens, and Crosslinking Reagents”, in Bioconjug.Chem., 3, 2013. As noted above, the overall linkage between themulti-armed polymer core and each drug molecule preferably comprises ahydrolytically degradable portion, such as an ester linkage, so that theactive agent is released over time from the multi-armed polymer core.

The multi-arm polymeric conjugates provided herein (as well as thecorresponding reactive polymer precursor molecules, and so forth)comprise a linker segment, Q, and a spacer segment, X. Exemplary spacersor linkers can include segments such as those independently selectedfrom the group consisting of —O—, —S—, —NH, —C(O)—, —O—C(O)—, —C(O)—O—,—C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—,—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—,—CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—, —CH₂—NH—CH₂—,—CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]₀₋₆—(OCH₂CH₂)₀₋₂—, —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, and—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—.

In any of the above examples, a simple cycloalkylene group, e.g. 1,3- or1,4-cyclohexylene, may replace any two, three or four carbon alkylenegroup. For purposes of the present disclosure, however, a series ofatoms is not a spacer moiety when the series of atoms is immediatelyadjacent to a water-soluble polymer segment and the series of atoms isbut another monomer, such that the proposed spacer moiety wouldrepresent a mere extension of the polymer chain. A spacer or linker asdescribed herein may also comprise a combination of any two or more ofthe above groups, in any orientation.

Referring to structure I, Q is a linker, preferably one that ishydrolytically stable. Typically, Q contains at least one heteroatomsuch as O, or S, or NH, where the atom proximal to R in Q, when takentogether with R, typically represents a residue of the core organicradical R. Generally, Q contains from 1 to about 10 atoms, or from 1 toabout 5 atoms. Q typically contains one of the following numbers ofatoms: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Illustrative Qs include O, S,or —NH—C(O)—.

Again in reference to structure I, X is a spacer that comprises ahydrolyzable linkage, where the hydrolyzable linkage is attacheddirectly to the active agent, D. Typically, at least one atom of thehydrolyzable linkage is contained in the active agent in its unmodifiedform, such that upon hydrolysis of the hydrolyzable linkage comprisedwithin X, the active agent, D, is released. Generally speaking, thespacer has an atom length of from about 4 atoms to about 50 atoms, ormore preferably from about 5 atoms to about 25 atoms, or even morepreferably from about 5 atoms to about 20 atoms. Typically, the spaceris of an atom length selected from the group consisting of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. When consideringatom chain length, only atoms contributing to the overall distance areconsidered. For example, a spacer having the structure, —CH₂—C(O)—NH—CH₂CH₂ O—CH₂ CH₂ O—C(O)—O— has a chain length of 11 atoms, sincesubstituents are not considered to contribute significantly to thelength of the spacer.

In yet another particular embodiment, X possesses the structure: Y—Z,where Y is a spacer fragment covalently attached to Z, a hydrolyticallydegradable linkage. In certain embodiments, Z itself may not constitutea hydrolytically degradable linkage, however, when taken together withY, or at least a portion of Y, forms a linkage that is hydrolyticallydegradable.

In yet a more particular embodiment of the spacer, X, Y has thestructure: —(CR_(x)R_(y))_(a)—K—(CR_(X)R_(y))_(b)—(CH₂CH₂O)_(c)—,wherein each R₁ and R₂, in each occurrence, is independently H or anorganic radical selected from the group consisting of alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,and substituted aryl, a ranges from 0 to 12 (i.e., can be 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12), b ranges from 0 to 12 (i.e., can be 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), K is selected from —C(O)—,—C(O)NH—, —NH—C(O)—, —O—, —S—, O—C(O)—, C(O)—O—, O—C(O)—O—, O—C(O)—NH—,NH—C(O)—O—, c ranges from 0 to 25, and Z is selected from C(O)—O—,O—C(O)—O—, —O—C(O)—NH—, and NH—C(O)—O—. The particular structure of Kand of Z will depend upon the values of each of a, b, and c, such thatnone of the following linkages result in the overall structure of spacerX: —O—O—, NH—O—, NH—NH—.

Preferably, Y comprises (—CH₂)_(a)—C(O)NH—(CH₂)_(0,1)—(CH₂CH₂O)₀₋₁₀.

In yet another embodiment of the spacer, X, Y has the structure:—(CR_(x)R_(y))_(a)—K—(CR_(x)R_(y))_(b)—(CH₂CH₂NH)_(c)—, where thevariables have the values previously described. In certain instances,the presence of the short ethylene oxide or ethyl amino fragments inspacer, X, can be useful in achieving good yields during preparation ofthe prodrug conjugate, since the presence of the linker can help tocircumvent problems associated with steric hindrance, due to themulti-armed reactive polymer, the structure of the active agent, or acombination of both. Preferably, c is selected from the group consistingof 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

Preferably, R_(x) and R_(y) in each occurrence are independently H orlower alkyl. In one embodiment, R_(x) and R_(y) are in each occurrenceH. In yet another embodiment, “a” ranges from 0 to 5, i.e., is selectedfrom 0, 1, 2, 3, 4, or 5. In yet another embodiment, b ranges from 0 to5, i.e., is selected from 0, 1, 2, 3, 4, or 5. In yet anotherembodiment, c ranges from 0 to 10. In yet another embodiment, K is—C(O)—NH. Any of the embodiments described herein is meant to apply notonly to generalized structure I, but also extend to particularcombinations of embodiments.

In yet another embodiment, R_(x) and R_(y) in each occurrence are H, ais 1, K is —C(O)—NH, and b is 0 or 1.

Particular examples of X include —CH₂—C(O)—NH—CH₂—C(O)O— (here, Ycorresponds to —CH₂—C(O)—NH—CH₂— and Z corresponds to —C(O)—O—), and—CH₂—C(O)—NH—(CH₂CH₂O)₂—C(O)—O— (here, Y corresponds to—CH₂—C(O)—NH—(CH₂CH₂O)₂— and Z corresponds to —C(O)—O—).

The Polymer, POLY₁

In structure I, POLY₁ represents a water-soluble and non-peptidicpolymer. POLY₁ in each polymer arm of structure I is independentlyselected, although preferably, each polymer arm will comprise the samepolymer. Preferably, each of the arms (i.e., each “(-Q-POLY₁-X-D) ofstructure I is identical. Any of a variety of polymers that arenon-peptidic and water-soluble can be used to form a conjugate inaccordance with the present invention. Examples of suitable polymersinclude, but are not limited to, poly(alkylene glycols), copolymers ofethylene glycol and propylene glycol, poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(acrylic acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384, which is incorporated by reference herein in its entirety,and copolymers, terpolymers, and mixtures of any one or more of theabove.

Preferably, POLY₁ is a polyethylene glycol or PEG. POLY₁ can be in anyof a number of geometries or forms, including linear chains, branched,forked, etc., although preferably POLY₁ is linear (i.e., in each arm ofthe overall multi-arm structure) or forked. A preferred structure for amulti-armed polymer prodrug having a “forked” polymer configuration isas follows:

F represents a forking group, and the remaining variables are aspreviously described. Preferably, the fork point in the forking group,F, comprises or is (—CH), though it may also be a nitrogen atom (N). Inthis way, each polymer arm is forked to possess two active agentmoieties releasably covalently attached thereto, rather than one.

Illustrative forked polymers useful for preparing a multi-armed polymerof the type shown in Fig. XII are described in U.S. Pat. No. 6,362,254.

When POLY₁ is PEG, its structure typically comprises —(CH₂CH₂O)_(n)—,where n ranges from about 5 to about 400, preferably from about 10 toabout 350, or from about 20 to about 300.

In the multi-arm embodiments described here, each polymer arm, POLY₁,typically has a molecular weight corresponding to one of the following:200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 7500, 8000, 9000, 10000, 12,000, 15000, 17,500,18,000, 19,000, 20,000 daltons or greater. Overall molecular weights forthe multi-armed polymer configurations described herein (that is to say,the molecular weight of the multi-armed polymer as a whole) generallycorrespond to one of the following: 800, 1000, 1200, 1600, 2000, 2400,2800, 3200, 3600, 4000, 5000, 6000, 8000, 10,000, 12,000, 15,000,16,000, 20,000, 24,000, 25,000, 28,000, 30,000, 32,000, 36,000, 40,000,45,000, 48,000, 50,000, 60,000, 80,000 or greater.

Typically, the overall molecular weight for a multi-armed polymer of theinvention ranges from about 800 to about 60,000 daltons. Other preferredmolecular weight ranges for a multi-armed polymer of the invention arefrom about 1,000 to about 40,000 daltons, or from about 5,000 to about30,000 daltons, or even from about 20,000 to about 80,000 daltons.

Active Agent, D.

Returning now to structure I, D is an active agent moiety, and q (thenumber of independent polymer arms) ranges from about 3 to about 50.Preferably, q ranges from about 3 to about 25. More preferably, q isfrom 3 to about 10, and possesses a value of 3, 4, 5, 6, 7, 8, 9, or 10.The active agent moiety, D contains at least one functional groupsuitable for covalent attachment to the multi-armed polymer describedherein to form a hydrolyzable linkage, such that upon hydrolysis, theactive agent is released in its unmodified form.

In accordance with one embodiment of the invention, a prodrug conjugateis characterized as a polymer having from about 3 to about 25 activeagent molecules covalently attached thereto. More particularly, theconjugate is characterized as a water-soluble polymer having 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, oractive agent molecules covalently attached thereto. In a furtherembodiment, the conjugate of the invention has from about 3 to about 8active agent molecules covalently attached to the water-soluble polymer.Typically, although not necessarily, the number of polymer arms willcorrespond to the number of active agents covalently attached to thewater-soluble polymer. That is to say, in the case of a polymer having acertain number of polymer arms (e.g., X), each having a reactivefunctional group at its terminus, the number of active agents covalentlyattached thereto in the resulting conjugate is preferably X.

In yet another embodiment, rather than having multiple polymer armsemanating from a central organic radical core, a conjugate of theinvention is characterized as a water-soluble polymer having pendantactive agent moieties covalently attached thereto, each preferablycovalently attached by a degradable linkage. In such an embodiment, thestructure of the polymer prodrug conjugate is described generally asPOLY₁(X-D)_(q), where and POLY₁, X, D, and q are as set forth above, andthe polymer, typically a linear polymer, possesses “q” active agentmoieties attached thereto, typically at discrete lengths along thepolymer chain, via the spacer X which contains a hydrolyzable linkage.

In a specific embodiment, the active agent moiety is a small moleculepossessing a molecular weight of less than about 1000. In yet additionalembodiments, the small molecule drug possesses a molecular weight ofless than about 800, or even less than about 750. In yet anotherembodiment, the small molecule drug possesses a molecular weight of lessthan about 500 or, in some instances, even less than about 300.

Preferred active agent moieties include anticancer agents. Particularlypreferred are oncolytics having at least one hydroxyl group.

One preferred class of active agents is the camptothecins. In oneembodiment, a camptothecin for use in the invention corresponds to thestructure:

wherein R₁-R₅ are each independently selected from the group consistingof hydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxcarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or

R₂ together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy;

R₆ is H or OR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl; and

L is the site of attachment to X.

The term “camptothecin compound” as used herein includes the plantalkaloid 20(S)-camptothecin, as well as pharmaceutically activederivatives, analogues and metabolites thereof. Examples of camptothecinderivatives include, but are not limited to, 9-nitro-20(S)-camptothecin,9-amino-20(S)-camptothecin, 9-methyl-camptothecin,9-chloro-camptothecin, 9-fluoro-camptothecin, 7-ethyl camptothecin,10-methyl-camptothecin, 10-chloro-camptothecin, 10-bromo-camptothecin,10-fluoro-camptothecin, 9-methoxy-camptothecin, 11-fluoro-camptothecin,7-ethyl-10-hydroxy camptothecin (SN38), 10,11-methylenedioxycamptothecin, and 10,11-ethylenedioxy camptothecin, and7-(4-methylpiperazinomethylene)-10,11-methylenedioxy camptothecin,7-ethyl-10-(4-(1-piperidino)-1-piperidino)-carbonyloxy-camptothecin,9-hydroxy-camptothecin, and 11-hydroxy-camptothecin. Particularlypreferred camptothecin compounds include camptothecin, irinotecan, andtopotecan.

Native and unsubstituted, the plant alkaloid camptothecin can beobtained by purification of the natural extract, or may be obtained fromthe Stehlin Foundation for Cancer Research (Houston, Tex.). Substitutedcamptothecins can be obtained using methods known in the literature orcan be obtained from commercial suppliers. For example,9-nitro-camptothecin may be obtained from SuperGen, Inc. (San Ramon,Calif.), and 9-amino-camptothecin may be obtained from IdecPharmaceuticals (San Diego, Calif.). Camptothecin and various analoguesand derivatives may also be obtained from standard fine chemical supplyhouses, such as Sigma Chemicals.

Certain preferred camptothecin compounds correspond to the generalizedstructure below.

wherein R₁-R₅ are each independently selected from the group consistingof hydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxcarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or

R₂ together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy; and

R₆ is H or OR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl.

Exemplary substituting groups include hydroxyl, amino, substitutedamino, halo, alkoxy, alkyl, cyano, nitro, hydroxycarbonyl,alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonylamino, aryl (e.g.,phenyl), heterocycle, and glycosyl groups.

In one particularly preferred embodiment, D is irinotecan, where the Hon the 20-position hydroxyl is absent in the final multi-armed prodrugconjugate.

In yet another embodiment, D is paclitaxel or docetaxel. Oneparticularly preferred D is docetaxel, where the H at the 2′ position isabsent in the final multi-armed polymer conjugate:

Active agents for use in the invention also include hypnotics andsedatives, psychic energizers, tranquilizers, respiratory drugs,anticonvulsants, muscle relaxants, antiparkinson agents (dopamineantagnonists), analgesics, anti-inflammatories, antianxiety drugs(anxiolytics), appetite suppressants, antimigraine agents, musclecontractants, anti-infectives (antibiotics, antivirals, antifungals,vaccines) antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxicants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

More particularly, the active agent may fall into one of a number ofstructural classes, including but not limited to small molecules,oligopeptides, polypeptides or protein mimetics, fragments, oranalogues, steroids, nucleotides, oligonucleotides, electrolytes, andthe like. Preferably, an active agent for use in the invention possessesa free hydroxyl, carboxyl, thio, amino group, or the like (i.e.,“handle”) suitable for covalent attachment to the polymer. Preferably,an active agent possesses at least one functional group suitable forforming a hydrolyzable linkage when reacted with a multi-armed polymerprecursor suitable for forming a prodrug conjugate of the invention.

Alternatively, the drug is modified by introduction of a suitable“handle”, preferably by conversion of one of its existing functionalgroups to a functional group suitable for formation of a hydrolyzablecovalent linkage between the multi-armed polymer and the drug. Ideally,such a modification should not adversely impact the therapeutic effector activity of the active agent to a significant degree. That is to say,any modification of an active agent to facilitate its attachment to amulti-armed polymer of the invention should result in no greater thanabout a 30% reduction of its bioactivity relative to the known parentactive agent prior to modification. More preferably, any modification ofan active agent to facilitate its attachment to a multi-armed polymer ofthe invention preferably results in a reduction of its activity relativeto the known parent active agent prior to modification of no greaterthan about 25%, 20%, 15%, 10% or 5%.

Specific examples of active agents include proteins, small moleculemimetics thereof, and active fragments (including variants) of thefollowing: aspariginase, amdoxovir (DAPD), antide, becaplermin,calcitonins, cyanovirin, denileukin diftitox, erythropoietin (EPO), EPOagonists (e.g., peptides from about 10-40 amino acids in length andcomprising a particular core sequence as described in WO 96/40749),dornase alpha, erythropoiesis stimulating protein (NESP), coagulationfactors such as Factor V, Factor VII, Factor VIIa, Factor VIII, FactorIX, Factor X, Factor XII, Factor XIII, von Willebrand factor; ceredase,cerezyme, alpha-glucosidase, collagen, cyclosporin, alpha defensins,beta defensins, exedin-4, granulocyte colony stimulating factor (GCSF),thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), fibrinogen,filgrastim, growth hormones human growth hormone (hGH), growth hormonereleasing hormone (GHRH), GRO-beta, GRO-beta antibody, bone morphogenicproteins such as bone morphogenic protein-2, bone morphogenic protein-6,OP-1; acidic fibroblast growth factor, basic fibroblast growth factor,CD-40 ligand, heparin, human serum albumin, low molecular weight heparin(LMWH), interferons such as interferon alpha, interferon beta,interferon gamma, interferon omega, interferon tau, consensusinterferon; interleukins and interleukin receptors such as interleukin-1receptor, interleukin-2, interluekin-2 fusion proteins, interleukin-1receptor antagonist, interleukin-3, interleukin-4, interleukin-4receptor, interleukin-6, interleukin-8, interleukin-12, interleukin-13receptor, interleukin-17 receptor; lactoferrin and lactoferrinfragments, luteinizing hormone releasing hormone (LHRH), insulin,pro-insulin, insulin analogues (e.g., mono-acylated insulin as describedin U.S. Pat. No. 5,922,675), amylin, C-peptide, somatostatin,somatostatin analogs including octreotide, vasopressin, folliclestimulating hormone (FSH), influenza vaccine, insulin-like growth factor(IGF), insulintropin, macrophage colony stimulating factor (M-CSF),plasminogen activators such as alteplase, urokinase, reteplase,streptokinase, pamiteplase, lanoteplase, and teneteplase; nerve growthfactor (NGF), osteoprotegerin, platelet-derived growth factor, tissuegrowth factors, transforming growth factor-1, vascular endothelialgrowth factor, leukemia inhibiting factor, keratinocyte growth factor(KGF), glial growth factor (GGF), T Cell receptors, CDmolecules/antigens, tumor necrosis factor (TNF), monocytechemoattractant protein-1, endothelial growth factors, parathyroidhormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha 1,thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosin beta 9,thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE) compounds,VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosphonates,respiratory syncytial virus antibody, cystic fibrosis transmembraneregulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), and anti-CMVantibody. Exemplary monoclonal antibodies include etanercept (a dimericfusion protein consisting of the extracellular ligand-binding portion ofthe human 75 kD TNF receptor linked to the Fc portion of IgG1),abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomabtiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,olizumab, rituximab, and trastuzumab (herceptin).

Additional agents suitable include but are not limited to amifostine,amiodarone, aminocaproic acid, aminohippurate sodium, aminoglutethimide,aminolevulinic acid, aminosalicylic acid, amsacrine, anagrelide,anastrozole, asparaginase, anthracyclines, bexarotene, bicalutamide,bleomycin, buserelin, busulfan, cabergoline, capecitabine, carboplatin,carmustine, chlorambucin, cilastatin sodium, cisplatin, cladribine,clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecins,13-cis retinoic acid, all trans retinoic acid; dacarbazine,dactinomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine,etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone,fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine,L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan,itraconazole, goserelin, letrozole, leucovorin, levamisole, lisinopril,lovothyroxine sodium, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, metaraminol bitartrate,methotrexate, metoclopramide, mexiletine, mitomycin, mitotane,mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, pilcamycin, porfimer, prednisone,procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus,streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide,testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine,vinorelbine, dolasetron, granisetron; formoterol, fluticasone,leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins,nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such aserythromycin, oleandomycin, troleandomycin, roxithromycin,clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin,josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin,andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, andstreptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, gramicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicllinase-sensitive agents like penicillin G, penicillin V;penicllinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefmetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, pentamidineisethiouate, albuterol sulfate, lidocaine, metaproterenol sulfate,beclomethasone diprepionate, triamcinolone acetamide, budesonideacetonide, fluticasone, ipratropium bromide, flunisolide, cromolynsodium, and ergotamine tartrate; taxanes such as paclitaxel; SN-38, andtyrphostines.

The above exemplary drugs are meant to encompass, where applicable,analogues, agonists, antagonists, inhibitors, isomers, polymorphs, andpharmaceutically acceptable salt forms thereof.

In yet another embodiment of the invention, the small molecule is nottaxol, or is not taxane-based.

Other preferred active agents for preparing a multi-armed polymerprodrug conjugate as described herein include platins, oxymorphoneanalogues, steroids, quinolones, isoquinolones, and fluoroquinolones,and nucleosides and nucleotides. Structures of illustrative compoundsbelonging to each of the above structural classes are provided below.

Compositions/Populations of Prodrug Conjugates

As stated above, in certain instances, a composition comprising amulti-arm polymer prodrug as described herein may comprise the prodrughaving one of more of its polymer arms absent drug, D. Such anoccurrence may arise, for example, due to incomplete reaction of themulti-armed reactive polymer with drug, D. Often, even in the instanceof favorable stoichiometry, i.e., using an excess of drug relative tothe number of reactive polymer arms, it can be difficult to drive thereaction to completion such that the product may comprise a mixturepolymer species.

In such instances, a composition of the invention may be described ascomprising a multi-arm polymer prodrug having the structureR(-Q-POLY₁-X′-D_(0,1))_(q). In the foregoing structure, R, Q, POLY₁, andq are as previously described. The variable, X′, is either (i) a spacer,X, comprising a hydrolyzable linkage, such that upon hydrolysis of thehydrolyzable linkage, D, is released, or is (ii) X″, a terminal moiety.The variable D is a small molecule, where D₁ indicates the presence of Dand D₀ indicates its absence. In reference to the foregoing structure,R(-Q-POLY₁-X′-D_(0,1))_(q), the following conditions also apply: if X′is X, then D is D₁, and if X′ is X″, then D is D₀. Typically, thecomposition comprises at least one multi-arm polymer prodrug specieswherein X′ is X. The terminal moiety, X″, typically corresponds to anunreacted functional group present on one or more arms of themulti-armed polymer, or, is an inert derivative thereof, e.g., resultingfrom work-up, such as a hydrolysis product.

More particularly, a composition of the invention may comprise one ormore prodrug species having the structure:

R-(Q-POLY₁-X-D₁)_(m)(Q-POLY₁-X″)_(s).

In the above structure, m and s are each integers, where the sum of mand s equals q (the total number of polymer arms emanating from theorganic radical core), and where m and s each independently have valuesranging from 0 to q. Preferably, the value of s is not equal to q, butrather is less than q. Ideally, in the instance of quantitativesubstitution of active agent, s equals zero and m equals q.

As an illustration, for a multi-armed polymer having 4 polymer arms, theconjugate composition may comprise one or more of the following polymerprodrug species, where the last component represents unreacted startingmaterial or its equivalent resulting from work up of the reactionmixture: R-(Q-POLY₁-X-D₁)₄, R-(Q-POLY₁-X-D₁)₃(Q-POLY₁-X″),R-(Q-POLY₁-X-D₁)₂(Q-POLY₁-X″)₂, R-(Q-POLY₁-X-D₁)(Q-POLY₁-X″)₃, andR-(Q-POLY₁-X″)₄ Preferably, the latter species, R-(Q-POLY₁-X″)₄, isabsent from the conjugate composition, or, if present, is present inminimal amounts, e.g., less than about 5% by weight, preferably lessthan about 2% by weight.

Thus, for instance, a prodrug composition based upon a multi-armedpolymer in accordance with the invention having four polymer arms maycomprise a mixture of up to five different polymer species, and morepreferably of up to four different polymer species in the instance wherethe product composition is essentially absent unreacted polymer startingmaterial. That is to say, in one particular embodiment of the invention,the composition comprises a number of polymer prodrug speciescorresponding to the number of polymer arms, q.

In one embodiment, a composition of the invention is characterized byhaving an average number of drug molecules per multi-armed polymer thatranges from about 52% to about 94% or greater of the idealized value. Inan alternative embodiment, a composition comprising a population ofmulti-armed polymer conjugate species is characterized by having anaverage number of drug molecules per multi-armed polymer that rangesfrom about 57% to about 87% or greater of the idealized value. As anillustration, in an instance in which the multi-armed polymer containsfour polymer arms, the idealized value of number of covalently attacheddrug molecules per multi-armed polymer is four, and an average number ofdrug molecules per multi-armed polymer ranging from about 52% to about94% or greater of the idealized value corresponds to an average numberof D per multi-arm polymer ranging from about 2.1 to 3.75.Alternatively, when the multi-armed polymer contains four polymer arms,and the average number of drug molecules per multi-armed polymer rangesfrom about 57% to about 87% or greater of the idealized value, then theresulting composition possesses an average number of D per multi-armpolymer ranging from about 2.1 to 3.75.

An exemplary composition forming part of the invention comprises amixture of one or more prodrug species having the structure:

where O-Irinotecan corresponds to:

m+s=4, and n ranges from about 40 to 500.

Even more particularly, a preferred multi-armed polymer prodrugcomposition in accordance with the invention includes one or more of thefollowing conjugate species:

In yet another embodiment, the composition comprises each of the aboveconjugate prodrug species. Preferably, in particular for prodrugconjugates having a number of polymer arms ranging from about 3 to about8, the majority species present in the composition are those havingeither an idealized number of drug molecules attached to the polymercore (“q”) or those having a combination of (“q”) and (“q−1”) drugmolecules attached to the polymer core.

Yet another exemplary composition forming part of the inventioncomprises a mixture of one or more prodrug species having the structure:

where m+s=4, and n ranges from 40 to 500.

Even more particularly, a preferred multi-armed polymer prodrugcomposition in accordance with the invention includes one or more of thefollowing conjugate species:

where O-Docetaxel corresponds to:

Alternatively, and most preferably, a multi-arm polymer prodrug asprovided herein possesses active agent covalently attached to eachpolymer arm, such that essentially quantitative substitution of activeagent in each of the polymer arms has taken place. For example, for amulti-arm polymer having 3 reactive polymer arms, in the resultingmulti-arm polymer prodrug, each of the 3 polymer arms possesses activeagent covalently attached thereto. In such embodiment, a composition ofthe invention is characterized by having an average number of drugmolecules per multi-armed polymer that corresponds essentially to itsidealized value (i.e., is essentially 100% of its idealized value).

The invention is meant to encompass each prodrug described herein,whether described singly or as forming part of a prodrug composition.

Method of Forming a Multi-Armed Polymer Prodrug Conjugate

Multi-armed reactive polymers, such as those for preparing a prodrug ofthe invention can be readily prepared from commercially availablestarting materials in view of the guidance presented herein, coupledwith what is known in the art of chemical synthesis.

Hydroxyl-terminated multi-armed PEGs having either a pentaerythritolcore or a glycerol core are available from Nektar, Huntsville Ala. Suchmulti-armed PEGs can be used directly for coupling to active agentshaving, e.g., a carboxyl group in a position suitable for coupling,e.g., to provide a polymer prodrug having a hydrolyzable carboxyl esterbond. Alternatively, terminal hydroxyls present on a multi-armed polymerprecursor can be oxidized to terminal carboxyl groups, e.g., forcoupling to hydroxyls present on an active agent.

Alternatively, a multi-armed reactive polymer for preparing a prodrug ofthe invention may be synthetically prepared. For instance, any of anumber of suitable polyol core materials can be purchased from achemical supplier such as Aldrich (St. Louis, Mo.). The terminalhydroxyls of the polyol are first converted to their anionic form,using, for example, a strong base, to provide a site suitable forinitiating polymerization, followed by direct polymerization of monomersubunits, e.g., ethylene oxide, onto the core. Chain building is allowedto continue until a desired length of polymer chain is reached in eachof the arms, followed by terminating the reaction, e.g., by quenching.

In an alternative approach, an activated multi-armed polymer precursorto the prodrugs of the invention can be synthetically prepared by firstproviding a desired polyol core material, and reacting the polyol undersuitable conditions with a heterobifunctional PEG mesylate of a desiredlength, where the non-mesylate PEG terminus is optionally protected toprevent reaction with the polyol core. The resulting multi-armed polymerprecursor is then suitable for additional transformations or directcoupling to an active agent, following deprotection if necessary.

Multi-armed polymer precursors based on polyamino cores can be prepared,for example, by direct coupling to a polymer reagent activated with anacylating agent such as an NHS ester, a succinimidyl carbonate, a BTCester or the like, to provide multi-armed polymer precursors having anamide linker, Q. Alternatively, a core molecule having multiple aminogroups can be coupled with an aldehyde terminated polymer, such as aPEG, by reductive amination (using, for example, a reducing agent suchas sodium cyanoborohydride) to provide a multi-armed polymer precursorhaving an internal amine linker, Q.

Although the polymer PEG is described as a representative polymer in thesynthetic descriptions above, such approaches apply equally as well toother water-soluble polymers described herein.

The prodrugs of the invention can be formed using known chemicalcoupling techniques for covalent attachment of activated polymers, suchas an activated PEG, to a biologically active agent (See, for example,POLY(ETHYLENE GLYCOL) CHEMISTRY AND BIOLOGICAL APPLICATIONS, AmericanChemical Society, Washington, D.C. (1997)). Selection of suitablefunctional groups, linkers, protecting groups, and the like to achieve amulti-arm polymer prodrug in accordance with the invention, will depend,in part, on the functional groups on the active agent and on themulti-armed polymer starting material and will be apparent to oneskilled in the art, based upon the contents of the present disclosure.

A multi-armed polymer of the invention suitable for coupling to anactive agent or derivatized active agent will typically have a terminalfunctional group such as the following: N-succinimidyl carbonate (seee.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmannet al. Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J.19:1177 (1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem.179:301 (1978)), succinimidyl propionate and succinimidyl butanoate(see, e.g., Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al., Makromol. Chem. 180:1381 (1979), succinimidyl ester(see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see,e.g., U.S. Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al.Eur. J. Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem.13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343(1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461).

In turning now to one of the preferred classes of active agents, thecamptothecins, since the 20-hydroxyl group of the camptothecin compoundis sterically hindered, a single step conjugation reaction is difficultto accomplish in significant yields. As a result, a preferred method isto react the 20-hydroxyl group with a short linker or spacer moietycarrying a functional group suitable for reaction with a multi-armpolymer. Such an approach is applicable to many small molecules,particularly those having a site of covalent attachment that isinaccessible to an incoming reactive polymer. Preferred linkers includet-BOC-glycine or other amino acids having a protected amino group and anavailable carboxylic acid group (See Zalipsky et al., “Attachment ofDrugs to Polyethylene Glycols”, Eur. Polym. J., Vol. 19, No. 12, pp.1177-1183 (1983)). The carboxylic acid group reacts readily with the20-hydroxyl group in the presence of a coupling agent (e.g.,dicyclohexylcarbodiimide (DCC)) and a base catalyst (e.g.,dimethylaminopyridine (DMAP)). Thereafter, the amino protecting group,such as t-BOC(N-tert-butoxycarbonyl), is removed by treatment with theappropriate deprotecting agent (e.g., trifluoroacetic acid (TFA) in thecase of t-BOC). The free amino group is then reacted with a multi-arm orforked polymer bearing carboxylic acid groups in the presence of acoupling agent (e.g., hydroxybenzyltriazole (HOBT)) and a base (e.g.,DMAP).

In a preferred embodiment, the spacer moiety is derived from andcomprises an amino acid and has the structure HO—C(O)—CH(R″)—NH-Gpwherein R″ is H, C1-C6 alkyl, or substituted C1-C6alkyl and Gp is aprotecting group protecting the alpha-amino group of the amino acid.Typical labile protecting groups include t-BOC and FMOC(9-flourenylmethloxycarbonyl). t-BOC is stable at room temperature andeasily removed with dilute solutions of TFA and dichloromethane. FMOC isa base labile protecting group that is easily removed by concentratedsolutions of amines (usually 20-55% piperidine in N-methylpyrrolidone).Preferred amino acids include alanine, glycine, isoleucine, leucine,phenylalanine, and valine.

Other spacer moieties having an available carboxylic acid group or otherfunctional group reactive with a hydroxyl group and a protected aminogroup can also be used in lieu of the amino acids described above. Forexample, a spacer moiety having the structure HOOC-alkylene-NH-Gp may beemployed, where Gp is as described above and the alkylene chain is, forexample, about 1 to about 20 carbon atoms in length. Spacers comprisingshort —(CH₂CH₂O)_(c)— groups or (CH₂CH₂NH)_(c) groups are alsopreferred, where c varies from about 0 to about 25. More particularly, cpossesses a value selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,and 12.

In a particular embodiment exemplified in Example 1, conjugation isaccomplished by first reacting the camptothecin compound witht-BOC-glycine, followed by deprotection of the glycine amino group andcoupling of the glycine-modified camptothecin to a 4-arm PEG moleculecomprising a pentaerythritol core.

In an alternative approach exemplified in Example 8, a bifunctionalspacer comprising a number of —(CH₂CH₂O)— subunits is provided. Oneterminal functional group of the spacer is an acid chloride (—O—C(O)—Cl)suitable for reaction with an active agent hydroxyl group to form acarbonate ester (i.e., a hydrolyzable linkage), while the other terminalfunctional group is a protected amine. The bifunctional spacer iscoupled to irinotecan, in particular to the 20-position hydroxylthereof, in the presence of a coupling agent such as DMAP to provide apartially modified active agent. In the partially modified active agent,a hydrolyzable bond, Z, has been introduced, coupled to a spacer, Y′having a protected terminus, which upon deprotection, is suitable forreaction with an activated multi-armed polymer. The partially modifiedactive agent is then reacted with a multi-armed polymer precursor havinga reactive terminus suitable for coupling to an amine, to provide astable amide linkage as part of the overall linkage, X.

In yet another aspect, the invention encompasses a method that isparticularly suited to conjugation of a polymer moiety to an activeagent having multiple reactive groups. The method is particularly usefulin instances in which single site modification of a drug or active agentis desired.

Conventional synthetic techniques for preparing a single sitepolymer-modified active agent from an agent having multiple reactivegroups typically involve multi-step reactions requiring multipleselective protection/deprotection steps. Such reactions are oftenhampered by low yields and difficulty in separation of the desiredproduct(s). In an attempt to avoid use of a protected active agentprecursor, the inventors attempted direct coupling of the exemplarysmall molecule, docetaxel, to a multi-armed polymer. Not unexpectedly,the reaction (i) generated a significant amount of side products whenconventional coupling reagents such as dicyclohexylcarbodiimide anddimethylaminopyridine (DCC/DMAP) were utilized, and (ii) was alsodifficult to analyze and purify. See Example 14.

To overcome this problem, the inventors developed a coupling reactionthat suppressed undesired side-reactions and promoted formation of thedesired product in good yields and purity.

In particular, provided herein is a method for covalently attaching apolyethylene glycol polymer to an active agent. The method includes thesteps of (i) providing an active agent comprising a first functionalgroup selected from amino, hydroxyl, and carboxyl (and activatedequivalents thereof), and (ii) reacting the active agent with apolyethylene glycol comprising a second functional group that isreactive with the first functional group. The reaction is carried out inthe presence of a coupling reagent and4-(dimethylamino)-pyridinium-p-toluenesulfonate (DPTS) under conditionseffective to promote reaction between the first and second functionalgroups, to thereby form a polyethylene glycol-active agent conjugate.

Preferred coupling agents are carbodiimides. Representative couplingagents include those selected from the group consisting ofdicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC),N-tert-butyl-N′-methylcarbodiimide (TBMC), andN-tert-butyl-N′-ethylcarbodiimide (TBEC).

One particularly preferred coupling reagent isN,N′-diisopropylcarbodiimide.

The reaction is typically carried out in an organic solvent. Suitablesolvents include dichloromethane, chloroform, acetonitrile, andtetrahydrofuran.

Typically, the coupling reaction is carried out at a temperature rangingfrom about 0° C. to about 100° C. Preferably, the reaction is carriedout at room temperature (i.e., absent heating or cooling).

Generally, the amount of DPTS ranges from about 0.05 to about 0.75equivalents relative to the first functional group, more preferably,from about 0.10 to 0.60 equivalents relative to the first functionalgroup. The amount of the coupling reagent generally ranges from about1.25 to 5 equivalents relative to the first functional group.

Preferred second functional groups include amino, hydroxyl, andcarboxyl, and activated equivalents thereof, where of course, the firstand second functional groups are selected to react with each other.

Active agents suitable for use in the method include proteins,oligopeptides, polypeptides, small molecules, antibodies, nucleotides,oligonucleotides, and lipids. Preferably, the active agent is a smallmolecule comprising a first functional group that is either hydroxyl orcarboxyl. The active agent may, in certain instances, possess more thanone first functional group.

Particularly preferred small molecules include taxanes (e.g., docetaxelor paclitaxel) and camptothecins such as those described herein.

Preferably, the reaction between the first and second functional groupsresults in formation of an ester bond (for example, resulting fromreaction of a carboxylic acid or activated carboxylic acid with thehydroxyl group of an alcohol).

In the method, the polyethylene glycol may possess any of a number ofgeometries, e.g., linear, branched, forked, and multi-armed polyethyleneglycol. Preferably, the polyethylene glycol is a multi-armed polymerhaving from about 3 to about 25 arms. Preferred multi-armed polymersinclude those previously described.

In one embodiment of the method, the polyethylene glycol comprises fromabout 1 to about 10 of the second functional groups. In an even morespecific embodiment, the polyethylene glycol comprises a number ofsecond functional groups selected from 1, 2, 3, 4, 5, and 6.

In yet another embodiment of the method, the active agent comprises morethan one first functional group, and the method does not comprise aprotection step, such that the resulting conjugate product is modifiedat only a single “first functional group” site.

In yet another embodiment, the method is effective to result information of less than about 15% of an N-acyl urea side product, andpreferably, less than about 10% of an N-acyl urea side product, and evenmore preferably, less than about 5% of an N-acyl urea side product. SeeExample 15 in which the synthetic method results in formation of adocetaxel-polyethylene conjugate having polyethylene glycol covalentlyattached, via an ester linkage, to a single hydroxyl site (e.g., the 2′hydroxyl site) on docetaxel.

The prodrug product may be further purified. Methods of purification andisolation include precipitation followed by filtration and drying, aswell as chromatography. Suitable chromatographic methods include gelfiltration chromatography and ion exchange chromatography.

Pharmaceutical Compositions

The invention provides pharmaceutical formulations or compositions, bothfor veterinary and for human medical use, which comprise one or morepolymer prodrugs of the invention or a pharmaceutically acceptable saltthereof, with one or more pharmaceutically acceptable carriers, andoptionally any other therapeutic ingredients, stabilizers, or the like.The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and not undulydeleterious to the recipient thereof. The compositions of the inventionmay also include polymeric excipients/additives or carriers, e.g.,polyvinylpyrrolidones, derivatized celluloses such ashydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose, Ficolls (a polymeric sugar),hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin),polyethylene glycols, and pectin. The compositions may further includediluents, buffers, binders, disintegrants, thickeners, lubricants,preservatives (including antioxidants), flavoring agents, taste-maskingagents, inorganic salts (e.g., sodium chloride), antimicrobial agents(e.g., benzalkonium chloride), sweeteners, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”, andpluronics such as F68 and F88, available from BASF), sorbitan esters,lipids (e.g., phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines, fatty acids and fattyesters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA,zinc and other such suitable cations). Other pharmaceutical excipientsand/or additives suitable for use in the compositions according to theinvention are listed in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), and in the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andin “Handbook of Pharmaceutical Excipients”, Third Ed., Ed. A. H. Kibbe,Pharmaceutical Press, 2000.

The prodrugs of the invention may be formulated in compositionsincluding those suitable for oral, rectal, topical, nasal, ophthalmic,or parenteral (including intraperitoneal, intravenous, subcutaneous, orintramuscular injection) administration. The compositions mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing the active agent or compound (i.e., the prodrug)into association with a carrier that constitutes one or more accessoryingredients. In general, the compositions are prepared by bringing theactive compound into association with a liquid carrier to form asolution or a suspension, or alternatively, bring the active compoundinto association with formulation components suitable for forming asolid, optionally a particulate product, and then, if warranted, shapingthe product into a desired delivery form. Solid formulations of theinvention, when particulate, will typically comprise particles withsizes ranging from about 1 nanometer to about 500 microns. In general,for solid formulations intended for intravenous administration,particles will typically range from about 1 nm to about 10 microns indiameter. Particularly preferred are sterile, lyophilized compositionsthat are reconstituted in an aqueous vehicle prior to injection.

A preferred formulation is a solid formulation comprising the multi-armpolymer prodrug where the active agent, D, is irinotecan. The solidformulation comprises sorbitol and lactic acid, and is typically dilutedwith 5% dextrose injection or 0.9% sodium chloride injection prior tointravenous infusion.

The amount of polymer conjugate in the formulation will vary dependingupon the specific opioid antagonist employed, its activity in conjugatedform, the molecular weight of the conjugate, and other factors such asdosage form, target patient population, and other considerations, andwill generally be readily determined by one skilled in the art. Theamount of conjugate in the formulation will be that amount necessary todeliver a therapeutically effective amount of camptothecin compound to apatient in need thereof to achieve at least one of the therapeuticeffects associated with the camptothecin compound, e.g., treatment ofcancer. In practice, this will vary widely depending upon the particularconjugate, its activity, the severity of the condition to be treated,the patient population, the stability of the formulation, and the like.Compositions will generally contain anywhere from about 1% by weight toabout 99% by weight prodrug, typically from about 2% to about 95% byweight prodrug, and more typically from about 5% to 85% by weightprodrug, and will also depend upon the relative amounts ofexcipients/additives contained in the composition. More specifically,the composition will typically contain at least about one of thefollowing percentages of prodrug: 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,or more by weight.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,lozenges, and the like, each containing a predetermined amount of theactive agent as a powder or granules; or a suspension in an aqueousliquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, adraught, and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which is optionally mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent or dispersing agent. Molded tablets comprised with a suitablecarrier may be made by molding in a suitable machine.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose, to which may also beadded any accessory ingredient(s). Such accessory ingredients mayinclude flavorings, suitable preservatives, an agent to retardcrystallization of the sugar, and an agent to increase the solubility ofany other ingredient, such as polyhydric alcohol, for example, glycerolor sorbitol.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the prodrug conjugate, whichcan be formulated to be isotonic with the blood of the recipient.

Nasal spray formulations comprise purified aqueous solutions of theactive agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, or hydrogenated fats orhydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise the active compound dissolved or suspendedin one or more media such as mineral oil, petroleum, polyhydroxyalcohols or other bases used for topical formulations. The addition ofother accessory ingredients as noted above may be desirable.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired polymer conjugate or a saltthereof. The desired formulation may be placed in a small chamber andnebulized. Nebulization may be accomplished by compressed air or byultrasonic energy to form a plurality of liquid droplets or solidparticles comprising the conjugates or salts thereof.

Methods of Use

The multi-armed polymer prodrugs of the invention can be used to treator prevent any condition responsive to the unmodified active agent inany animal, particularly in mammals, including humans.

The prodrugs of the invention are particularly useful as anticanceragents, i.e., have been shown to be effective in significantly reducingthe growth of certain solid tumors as evidenced by representative lungand colon cancers in in-vivo studies, among others. In particular, theprodrugs of the invention have been shown to be nearly five times moreeffective at preventing the growth of human lung cancer tumors and humancolon cancer tumors than the corresponding anticancer agent per se, whenadministered at comparable doses over illustrative time periods rangingfrom 30 to 80 days.

Examples 6 and 16 illustrate the effectiveness of illustrative polymerprodrugs of the invention in the treatment of lung cancer, based uponin-vivo lung cancer model results. Example 6 provides a comparison ofcertain 4-arm polyethylene glycol irinotecan prodrugs with unmodifiedirinotecan in mouse xenograft studies. Mice having sizable lung tumorswere treated with different doses of prodrug, and lung tumor size wasthen plotted at various times after commencement of treatment. (SeeFIGS. 2 and 3). The rate of tumor growth was slowed significantly inmice treated with 4-arm PEG irinotecan when compared to mice treatedwith irinotecan per se, thereby illustrating the superiority of theprodrug approach of the present invention when compared to treatmentwith unmodified drug. Similar results are observed with 4-arm PEGdocetaxel as described in detail in Example 16. FIGS. 16A-C and FIG. 17illustrate the improved anti-tumor effect of a representative prodrug ofthe invention in comparison to unmodified antitumor agent. Moreover, inaddition to providing superior efficacy when compared to theirunmodified counterparts, the prodrugs provided herein are also lesstoxic when evaluated in animal models. For instance, the maximumtolerated dose (MTD) for 4-arm-PEG-gly-irino-20K was significantlyhigher than for irinotecan (FIG. 22). Additionally, the occurrenceand/or extent and/or severity of side-effects such as neutropenia anddiarrhea was reduced for 4-arm-PEG-gly-irino-20K when compared toirinotecan administered at equivalent doses (FIG. 23).

Similar results are provided in Examples 11 and 18 (in vivo breastcancer model), Examples 2 and 12 (in vivo colon cancer model), and inExample 17 (in vivo prostate cancer model). In some cases,administration of a prodrug of the invention not only resulted in anenhanced anti-tumor effect when compared to unmodified drug, butresulted in complete suppression of tumor growth in the animal modelemployed—thus indicating the superiority of the prodrugs and methodsprovided herein over unmodified oncolytic in treating various types ofcancer.

The multi-armed polymer prodrugs of the invention, in particular, thosewhere the small molecule drug is an anticancer agent such as acamptothecin compound as described herein or other oncolytic such asdocetaxel, are useful in treating breast cancer, ovarian cancer, coloncancer, colorectal cancer, prostate cancer, gastric cancer, malignantmelanoma, small cell lung cancer, non-small cell lung cancer, thyroidcancers, kidney cancer, cancer of the bile duct, brain cancer, cancer ofthe head and neck, lymphomas, leukemias, rhabdomyosarcoma,neuroblastoma, and the like. The prodrugs of the invention areparticularly effective in targeting and accumulating in solid tumors.The prodrugs are also useful in the treatment of HIV and other viruses.

Methods of treatment comprise administering to a mammal in need thereofa therapeutically effective amount of a composition or formulationcontaining a polymer prodrug of the invention.

A therapeutically effective dosage amount of any specific prodrug willvary from conjugate to conjugate, patient to patient, and will dependupon factors such as the condition of the patient, the activity of theparticular active agent employed, and the route of delivery.

For camptothecin-type active agents, dosages from about 0.5 to about 100mg camptothecin/kg body weight, preferably from about 10.0 to about 60mg/kg, are preferred. For taxane-type active agents, dosages of prodrugare administered in amounts ranging from about 5 to about 500 mg/m² perday, based upon the amount of the taxane moiety. When administeredconjointly with other pharmaceutically active agents, even less of theprodrug may be therapeutically effective. The range set above isillustrative and those skilled in the art will determine optimal dosingof the prodrug based on clinical experience and the particular treatmentindication.

Methods of treatment also include administering a therapeuticallyeffective amount of a composition or formulation containing a multi-armpolymer prodrug of an anticancer agent, e.g., a camptothecin or taxanecompound as described herein, in conjunction with a second anticanceragent. For example, in the treatment of colorectal cancer, a multi-armpolymer prodrug of a camptothecin or docetaxel type compound may beadministered in conjuction with chemotherapeutics such as 5-fluorouracilor leucovorin xeloda, or with agents such as avastin, Erbitux®(cetuximab), or Vectibix™ (panitumumab). In the treatment of breastcancer, therapy may include administration of a multi-arm polymerprodrug as described herein, optionally in combination with xeloda,paclitaxel, docetaxel, or abraxane. In treating lung cancer, therapy mayinclude, along with administration of a prodrug of the invention,administration of cis-platin, carboplatin, gemcitabine, alimpta, anddocetaxel (the latter in the instance in which the prodrug itself doesnot comprise docetaxel).

In an exemplary course of treatment, a multi-arm polymer prodrug, e.g.,4-arm-PEG-GLY-IRINO-20K, is administered to patients having colorectalcancer. One such patient population comprises patients having advancedcolorectal cancer. A therapeutically effective amount of a multi-armpolymer prodrug such as 4-arm-PEG-GLY-IRINO-20K is administered incombination with cetuximab an at initial loading dose, followed byweekly doses at a lower dosage of cetuximab. The efficacy and safety ofsuch treatment is evaluated against unmodified irinotecan similarlyadministered in conjunction with cetuximab. Typically, up to about fourdifferent dosage levels of prodrug are evaluated. A preferred endpointof the treatment regimen is progression free survival for a period ofabout several months or longer.

Preferably, camptothecin type prodrugs are administered in combinationwith 5-fluorouracil and folinic acid, as described in U.S. Pat. No.6,403,569.

The prodrug of the invention may be administered once or several times aday, preferably once a day or less. Illustrative dosing schedulesinclude once per week, once every two weeks, or once every three weeks.In the instance of a maintenance dose, dosing may take place even lessfrequently than once every three weeks, such as once monthly. Theduration of the treatment may be once per day for a period of from twoto three weeks and may continue for a period of months or even years.The daily dose can be administered either by a single dose in the formof an individual dosage unit or several smaller dosage units or bymultiple administration of subdivided dosages at certain intervals.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

EXAMPLES

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis and the like, which arewithin the skill of the art. Such techniques are fully explained in theliterature. Reagents and materials are commercially available unlessspecifically stated to the contrary. See, for example, J. March,Advanced Organic Chemistry Reactions Mechanisms and Structure, 4th Ed.(New York: Wiley-Interscience, 1992), supra, and Comprehensive OrganicFunctional Group Transformations II, Volumes 1-7, Second Ed.: AComprehensive Review of the Synthetic Literature 1995-2003 (OrganicChemistry Series), Eds. Katritsky, A. R., et al., Elsevier Science.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C. and pressure is at ornear atmospheric pressure at sea level.

Although other abbreviations known by one having ordinary skill in theart will be referenced, other reagents and materials will be used, andother methods known by one having ordinary skill in the art will beused, the following list and methods description is provided for thesake of convenience.

ABBREVIATIONS

-   -   CM carboxymethyl or carboxymethylene (—CH₂COOH)    -   DCC 1,3-dicyclohexylcarbodiimide    -   DCM methylene chloride    -   DIC N,N′-diisopropylcarbodiimide    -   DPTS 4-(dimethylamino)-pyridinium-p-toluenesulfonate    -   DMF dimethylformamide    -   DMAP 4-(N,N-dimethylamino)pyridine    -   DMSO dimethyl sulfoxide    -   DI deionized    -   HCl hydrochloric acid    -   HOBT hydroxybenzyltriazole    -   HPLC high performance liquid chromatography    -   IPA isopropyl alcohol    -   K or kDa kilodaltons    -   MALDI-TOF Matrix Assisted Laser Desorption Ionization        Time-of-Flight    -   MeOH methanol    -   MW molecular weight    -   NMR nuclear magnetic resonance    -   RT room temperature    -   SCM succinimidylcarboxymethyl (—CH₂—COO—N-succinimidyl)    -   SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel        electrophoresis    -   SEC size exclusion chromatography    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran    -   TLC thin layer chromatography

Materials and Methods

Irinotecan was purchased from JiangSu HengRui Medicine Co. Ltd. (China).

Docetaxel (Taxotere®) was purchased from Hangzhou HETD Pharm & Chem Co.,Ltd. CHINA.

4-Arm-PEG_(20K)-CM and 4-arm-PEG_(20K)-SCM were prepared from4-arm-PEG_(20K)-OH (Nektar, Huntsville, Ala.).

Sources of the following reagents were as follows: Glycine tert-butylester (98%, Aldrich); 4-dimethylaminopyridine (DMAP, 99%, Aldrich);N,N′-diisopropylcarbodiimide (DIC, 99%, Acros),N,N′-dicyclohexylcarbodiimide (DCC, 99%, Acros),N,N-diisopropylethylamine (DIPEA, 99%, Aldrich), and p-toluenesulfonicacid (PTSA, 98.5%, Aldrich), and all reagents were used as received.Solvents were dried before use.

All ¹HNMR data was generated by a 300 or 400 MHz NMR spectrometermanufactured by Bruker.

Example 1 Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino Acetate Linked-Irinotecan)-20K

The product was synthesized via a three-step process using as startingmaterials irinotecan hydrochloride and 4-arm polyethylene glycolsuccinimidyl acetate. The yields at each step were typically greaterthan about 90%.

A. Synthesis of t-Boc-Glycine-Irinotecan

In a flask, 0.1 g Irinotecan (0.1704 mmoles), 0.059 g t-Boc-Glycine(0.3408 mmoles), and 0.021 g DMAP (0.1704 mmoles) were dissolved in 13mL of anhydrous dichloromethane (DCM). To the solution was added 0.070 gDCC (0.3408 mmoles) dissolved in 2 mL of anhydrous DCM. The solution wasstirred overnight at room temperature. The solid was removed through acoarse frit, and the solution was washed with 10 mL of 0.1N HCL in aseparatory funnel. The organic phase was further washed with 10 mL ofdeionized H₂O in a separatory funnel and then dried with Na₂SO₄. Thesolvent was removed using rotary evaporation and the product was furtherdried under vacuum. ¹H NMR (DMSO): δ 0.919 (t, CH₂CH ₃), 1.34 (s,C(CH₃)₃), 3.83 (m, CH₂), 7.66 (d, aromatic H).

B. Deprotection of t-Boc-Glycine-Irinotecan

0.1 g t-Boc-Glycine-Irinotecan (0.137 mmoles) was dissolved in 7 mL ofanhydrous DCM. To the solution was added 0.53 mL trifluoroacetic acid(TFA, 6.85 mmoles). The solution was stirred at room temperature underargon for 1 hour. The solvent was removed using rotary evaporation. Thecrude product was dissolved in 0.1 mL MeOH and then precipitated in 25mL of ether. The suspension was stirred in an ice bath for 30 minutes.The product, a pale to dark yellow solid, was collected by filtrationand dried under vacuum. ¹H NMR (DMSO): δ 0.92 (t, CH₂CH ₃), 1.29 (t,CH₂CH ₃), 5.55 (s, 2H), 7.25 (s, aromatic H).

C. Covalent Attachment of a Multi-Armed Activated Polymer to GlycineIrinotecan.

A. 0.516 g Glycine-Irinotecan (0.976 mmoles), 3.904 g 4arm-PEG(20K)-CM(0.1952 mmoles), 0.0596 g 4-(dimethylamino)pyridine (DMAP, 0.488mmoles), and 0.0658 g 2-hydroxybenzyltriazole (HOBT, 0.488 mmoles) weredissolved in 60 mL anhydrous methylene chloride. To the resultingsolution was added 0.282 g 1,3-dicyclohexylcarbodiimide (DCC, 1.3664mmoles). The reaction mixture was stirred under argon overnight at roomtemperature. The mixture was filtered through a coarse frit and thesolvent was removed using rotary evaporation. The syrup was precipitatedin 200 mL of cold isopropanol over an ice bath. The solid was filteredand then dried under vacuum. Yield: 4.08 g. ¹H NMR (DMSO): δ 0.909 (t,CH₂CH ₃), 1.28 (m, CH₂CH ₃), 3.5 (br m, PEG), 3.92 (s, CH₂), 5.50 (s,2H).

The structure of the product was further confirmed by ¹³C-NMR, FTIR, andMALDI. The molecular weight of the conjugate was confirmed by MALDI tobe approximately 22,000 g/mol.

Synonyms for the product include: (a) 20-pentaerythritolpoly(oxy-1,2,-ethanediyl)-carboxymethyl-glycinate-7-ethyl-10-hydroxycamptothecine10-[1,4′-bipiperidine]-1′-carboxylate; (b) 4-arm branched poly (ethyleneglycol) conjugate of(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3′,4′:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4′-bipiperidine]-1′-carboxylate;(c) irinotecan 20-O-ester of PEG20 KDA-glycine, (d)PEG20K-gly-irinotecan, (e) PEG20K-irinotecan, (f) 4-armPEG20K-gly-irinotecan, (g) PEG-Irinotecan, (h) 20-pentaerythritolpoly(oxy-1,2-ethanediyl)-carboxymethyl-glycinate-irinotecan; (i)irinotecan tetra 20-O-ester of PEG20 KDA-glycine, (j)4-arm-PEG-gly-irino-20K.

Although reaction scheme IX depicts PEG-irinotecan as having a discretemolecular weight and complete polymer loading, i.e., an irinotecan drugmolecule attached to each arm of the 4-arm polymer core, more plausibly,upon reaction, the polymer (having a molecular weight distribution)produces a prodrug having an average of 2.3 to 3.0 molecules ofirinotecan per 4-armed polymer core. That is to say, based uponirinotecan drug loading calculations, the product is a mixture of4-armed polymer where the polymer core has one irinotecan moleculeattached, two irinotecan molecules attached, three irinotecan moleculesattached, and four irinotecan molecules attached, and perhaps even nodrug attached, to provide a composition which consistently averages 2.3to 3 molecules of irinotecan per polymer.

Purity of the product, based upon analyses of different product lots,was 97.9±0.9%. Free irinotecan in the final product was typically 0.5%or lower.

B. The reaction described above was carried out essentially as describedabove with the exception that the multi-arm PEG reagent, 4-arm-PEG-SCM,was added as a solid to a methylene chloride solution ofglycine-irinotecan. Characterization of the product revealed essentiallycomplete conversion (essentially 100% conversion) of all ˜PEG-SCM groupsin the multi-arm polymer reagent to ˜PEG-glycine-irinotecan to providethe desired product, 4-arm-PEG-GLY-IRINO-20k.

Example 2 Anti-Tumor Activity ofPentaerythritolyl-4-Arm-(PEG-1-Methylene-2 Oxo-Vinylamino AcetateLinked-Irinotecan)-20K, “4-Arm-PEG-Gly-Irino-20K” in a Colon CancerMouse Xenograft Model

Human HT29 colon tumor xenografts were subcutaneously implanted inathymic nude mice. After about two weeks of adequate tumor growth (100to 250 mg), these animals were divided into different groups of ten miceeach. One group was dosed with normal saline (control), a second groupwas dosed with 60 mg/kg of irinotecan, and the third group was dosedwith 60 mg/kg of the 4-arm PEG-GLY-Irino-20K (dose calculated peririnotecan content). Doses were administered intravenously, with onedose administered every 4 days for a total of 3 administered doses. Themice were observed daily and the tumors were measured with caliperstwice a week. FIG. 1 shows the effect of irinotecan and PEG-irinotecantreatment on HT29 colon tumors in athymic nude mice.

As can be seen from the results depicted in FIG. 1, mice treated withboth irinotecan and 4-arm-PEG-GLY-Irino-20K exhibited a delay in tumorgrowth (anti-tumor activity) that was significantly improved whencompared to the control. Moreover, the delay in tumor growth wassignificantly better for the 4-arm-PEG-GLY-Irino-20K group of mice whencompared to the group of animals administered unconjugated irinotecan.

Example 3 Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino Acetate Linked-Irinotecan)-40K, “4-Arm-PEG-Gly-Irino-40K”

4-arm-PEG-GLY-IRINO-40K was prepared in an identical fashion to thatdescribed for the 20K compound in Example 1, with the exception that instep C, the multi-armed activated PEG reagent employed was 4arm-PEG(40K)-CM rather than the 20K material.

In a 2L round bottom reactor, 4-arm-PEG_(40k)-SCM (240 g) was dissolvedin 1.0 L anhydrous methylene chloride. To a separate 500 mL round bottomreactor, glycine-irinotecan TFA salt (1.0 equiv, 20 g) was dissolved in53 mL DMF and treated with 8.8 mL TEA, stirred at room temperature for 5minutes. Then the solution was added to the solution of4-arm-PEG_(40k)-SCM in methylene chloride. The reaction was stirred atRT for 15 hrs and then precipitated in 6 L Et₂O and filtered to isolatesolid product, which was dissolved in 2.0 L IPA and 200 ml methanol at60° C. in a 5L round bottom reactor. While stirred by a mechanicalstirrer, the solution was cooled to RT for the product to precipitateout, followed by filtering to give 4-arm-PEG_(40k)-glycine-irinotecan(241 g, drug content 4.3% based on HPLC analysis, yield based on drug,67%)

Example 4 Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino Acetate Linked-SN-38)-20K, “4-Arm-PEG-Gly-SN-38-20K”

4-arm PEG-GLY-SN-38-20K was prepared in a similar fashion to itsirinotecan counterpart as described in Example 1, with the exceptionthat the active agent employed was SN-38, an active metabolite ofcamptothecin, rather than irinotecan, where the phenolic-OH of SN-38 wasprotected with MEMCl (2-methoxyethoxymethyl chloride) during thechemical transformations, followed by deprotection with TEA to providethe desired multi-armed conjugate.

4-arm-PEG_(20k)-SCM (12.2 g) was dissolved in 100 mL CH₂Cl₂.(20)-glycine-SN38 TFA salt (1.4 g, 2.44 mmol) was dissolved in 20 mL DMFand treated with 0.38 mL TEA, then added to the solution of4-arm-PEG_(20k)-SCM. The reaction was stirred at RT for 18 hrs and thenprecipitated in Et₂O to get solid product, which was dissolved in 100 mLIPA at 50° C., then cooled to RT to gave4-arm-PEG_(20k)-glycine-(20)-SN38 (12 g, drug content 6% based on HPLC).

Example 5 Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino Acetate Linked-SN-38)-40K, “4-Arm-PEG-Gly-SN-38-40K”

4-arm PEG-GLY-SN-38-40K was prepared in a similar fashion to the 20Kversion described above, with the exception that the multi-armedactivated PEG reagent employed was 4 arm-PEG(40K)-CM rather than the 20Kmaterial.

4-arm-PEG_(40k)-SCM (34.9 g) was dissolved in 200 mL CH₂Cl₂.(20)-Glycine-SN38 TFA salt (2.0 g, 3.49 mmol) was dissolved in 20 mL DMFand treated with 0.6 mL TEA, then added to the solution of4-arm-PEG_(20k)-SCM. The reaction was stirred at RT for 18 hrs and thenprecipitated in Et₂O to get solid product, which was dissolved in 100 mLIPA at 50° C., then cooled to RT to gave4-arm-PEG_(20k)-glycine-(20)-SN38 (34 g, drug content 2.3% based on HPLCanalysis).

Example 6 Additional Xenograft Studies—Lung and Colon Cancer Models

Additional mouse xenograft studies were conducted to further examine theefficacy of exemplary multi-armed polymer conjugates of the invention.

Athymic nude mice were implanted subcutaneously with human cancer celllines (lung cancer cell line NCI-H460, and colon cancer cell line HT-29)and the tumors allowed to grow to approximately 150 mg in size. Theanimals were divided into groups of ten mice each.

Various compounds and doses were evaluated as follows: irinotecan (40,60 and 90 mg/kg); 4-arm-PEG-GLY-IRINO-20K (40, 60, and 90 mg/kg);4-arm-PEG-GLY-IRINO-40K ((40, 60, and 90 mg/kg); 4-arm-PEG-GLY-SN-38-20K(7.5, 15, 30 mg/kg), and PEG-GLY-SN-38-40K (7.5, 15, 30 mg/kg). Doseswere administered intravenously, with one dose administered every 4 daysfor a total of 3 administered doses.

Tumor volume measurements were taken over a period of 60-80 days; tumorvolumes were converted to tumor weight. Body weights were also measuredover the same period to provide an indication of weight loss. Theresults are presented graphically in FIGS. 2-5.

Example 7 Pharmacokinetic Studies

A. Single Dose Colon Tumor Xenograft Study in Mice

A comparative single dose pharmacokinetic (PK) study of a multi-armedPEG-irinotecan versus unmodified irinotecan in nude mice was conductedto assess tumor distribution of parent and metabolite drug.

The study employed 108 nude mice, 36 mice per group, 4 animals persample point. Drug was administered intravenously as a single dose. Drugform and doses were as follows: irinotecan (40 mg/kg);4-arm-PEG-GLY-IRINO-20K (40 mg/kg equivalents); 4-arm-PEG-GLY-IRINO-40K((40 mg/kg equivalents). Venous plasma and tumor tissue samples weretaken at the following time points: 20 minutes, 40 minutes, and 1, 2, 4,12, 24, 48, and 72 hours, and evaluated for concentrations of thefollowing species: 4-arm-PEG-GLY-IRINO-20K, 4-arm-PEG-GLY-IRINO-40K,irinotecan and SN-38. The results are plotted in FIGS. 6 to 13.

As can be seen in FIGS. 6-13, based upon the rate of decline of themulti-armed PEGylated species in tumor tissue in comparison to plasma,the PEGylated species demonstrate a notable increase in tumor retentiontime when compared to unmodified parent drug.

In looking at the metabolite results, the concentrations of SN-38derived from the PEGylated compounds appear to be increasing at the endof the 72 hour period, while in contrast, SN-38 derived from irinotecanis essentially cleared in 12 hours. In sum, the tumor exposure to SN38following administration of either of the PEGylated compounds isapproximately five times greater than for irinotecan over the same 72hour sampling period. In sum, both multi-arm PEGylated compounds providean increased inhibition of tumor growth (colon and lung) for bothin-vivo tumor models investigated in comparison to unmodified drug. Morespecifically, both multi-arm PEGylated compounds demonstrated a markedsuppression of tumor growth when compared to unmodified drug in mousexenograft models, indicating the effectiveness of such compounds asanti-cancer agents. Lastly, administration of the multi-arm PEGylatedirinotecan compounds described herein appears to cause less diarrhea inrats than irinotecan itself.

B. Additional Single Dose Pharmacokinetic Studies

Single dose pharmacokinetic studies were additionally carried out inrats and dog (data not shown). In all species examined, dosing of4-arm-PEG-GLY-IRINO-20K resulted in sustained and greater exposure toirinotecan and SN38 than dosing with irinotecan per se.

Following dosing with 4-arm-PEG-GLY-IRINO-20K, SN38 t½ values wereapproximately one order of magnitude greater than those followingirinotecan dosing in both rodent and dogs. Differences in SN38 AUCvalues were in the range of 400- to 500-fold for rodents and 2- to6-fold in dogs. The PK of 4-arm-PEG-GLY-IRINO-20K was dominated by thedisposition properties of the PEG moiety. In the rodent and dog,PEG-Irinotecan had a plasma half-life of 2.7 to 5.9 hours and 23 to 92hours, respectively, low plasma clearance (CL) relative to hepatic bloodflow, and a small volume of distribution (Vd) relative to total bodywater volume. In contrast, irinotecan CL and Vd values were greater thanthose of PEG-Irinotecan. At pharmacologically relevant doses, t½ valuesfor irinotecan derived from 4-arm-PEG-GLY-IRINO-20K were about 61 to 68hours in the dog at 6 mg/kg (120 mg/m²), 123 hours in the rat at 20mg/kg (120 mg/m²), and 6 to 10 hours in the mouse at 40 mg/kg (120mg/m²). At pharmacologically relevant doses, t½ values for SN38 derivedfrom PEG-Irinotecan were 75 to 130 hours in the dog at 6 mg/kg (120mg/m²), 40 hours in the rat at 20 mg/kg (120 mg/m²), and 12 to 16 hoursin the mouse at 40 mg/kg (120 mg/m²). At pharmacologically relevantdoses of irinotecan, SN38 t½ values were about 3 to 7 hours and in ratsat 60 mg/kg and 3 to 10 hours in dogs at 6 mg/kg.

Interspecies differences in the metabolism of PEG-Irinotecan wereobserved (Table 1). SN38 reached high levels and had very high AUCs inrodents, presumably because of more rapid and extensive cleavage ofirinotecan due to higher level of esterases in rodent plasma than thatof dogs or humans. The systemic clearance of PEG-Irinotecan in ratsdecreased with increasing doses (4 to 20 mg/kg) suggestingdose-dependent kinetics for the drug in the rat. The systemic clearanceof PEG-Irinotecan in dogs was dose independent for the dose range of 6to 60 mg/kg.

TABLE 1 Species Comparisons of Single-dose PK Parameters FollowingPEG-Irinotecan Administration AUC Cmax t½ PEG- mouse < rat < dog mouse <rat < dog Dog > rat = Irinotecan mouse SN38 mouse >> rat >> dog Mouse >>rat >> dog Dog > rat > mouse

In humans, a clearance rate of 1.82 mL/hr/kg and a half-life of 83 hoursare predicted for 4-arm-PEG-GLY-IRINO-20K.

Example 8 Synthesis ofPentaerythritolyl-4-Arm-(PEG-2-{2-[2-1-Hydroxy-2-Oxo-Vinyloxy)-Ethoxy]-Ethylamino}-Propen-1-OneLinked—Irinotecan)-20K and -40K

A. 2-(2-t-Boc-aminoethoxy)ethanol (1)

2-(2-Aminoethoxy)ethanol (10.5 g, 0.1 mol) and NaHCO₃ (12.6 g, 0.15 mol)were added to 100 mL CH₂Cl₂ and 100 mL H₂O. The solution was stirred atRT for 10 minutes, then di-tert-butyl dicarbonate (21.8 g, 0.1 mol) wasadded. The resulting solution was stirred at RT overnight, thenextracted with CH₂Cl₂ (3×100 mL). The organic phases were combined anddried over anhydrous sodium sulfate and evaporated under vacuum. Theresidue was subjected to silica gel column chromatography(CH₂Cl₂:CH₃OH=50:1˜10:1) to afford 2-(2-t-Boc-aminoethoxy)ethanol (1)(16.0 g, 78 mmol, yield 78%).

B. 2-(2-t-Boc-aminoethoxy)ethoxycarbonyl-Irinotecan (2)

2-(2-t-Boc-aminoethoxy)ethanol (1) (12.3 g, 60 mmol) and4-dimethylaminopyridine (DMAP) (14.6 g, 120 mmol) were dissolved in 200ml anhydrous CH₂Cl₂. Triphosgene (5.91 g, 20 mmol) was added to thesolution while stirring at room temperature. After 20 minutes, thesolution was added to a solution of irinotecan (6.0 g, 10.2 mmol) andDMAP (12.2 g, 100 mmol) in anhydrous CH₂Cl₂ (200 mL). The reaction wasstirred at RT for 2 hrs, then washed with HCl solution (pH=3, 2L) toremove DMAP. The organic phases were combined and dried over anhydroussodium sulfate. The dried solution was evaporated under vacuum andsubjected to silica gel column chromatography (CH₂Cl₂:CH₃OH=40:1˜10:1)to afford 2-(2-t-Boc-aminoethoxy)ethoxycarbonyl-irinotecan (2) (4.9 g,6.0 mmol, yield 59%).

C. 2-(2-aminoethoxy)ethoxycarbonyl-irinotecan TFA salt (3)

2-(2-t-Boc-aminoethoxy)ethoxycarbonyl-irinotecan (2) (4.7 g, 5.75 mmol)was dissolved in 60 mL CH₂Cl₂, and trifluoroacetic acid (TFA) (20 mL)was added at RT. The reaction solution was stirred for 2 hours. Thesolvents were removed under vacuum and the residue was added to ethylether and filtered to give a yellow solid as product 3 (4.3 g, yield90%).

D. 4-arm-PEG_(20k)-carbonate-inotecan (4)

4-arm-PEG_(20k)-SCM (16.0 g) was dissolved in 200 mL CH₂Cl₂.2-(2-aminoethoxy)ethoxycarbonyl-irinotecan TFA salt (3) (2.85 g, 3.44mmol) was dissolved in 12 mL DMF and treated with 0.6 mL TEA, then addedto a solution of 4-arm-PEG_(20k)-SCM. The reaction was stirred at RT for12 hrs then precipitated in Et₂O to yield a solid product, which wasdissolved in 500 mL IPA at 50° C. The solution was cooled to RT and theresulting precipitate collected by filtration to give4-arm-PEG_(20k)-glycine-irinotecan (4) (16.2 g, drug content 7.5% basedon HPLC analysis). Yield: 60%.

E. 4-arm-PEG_(40k)-carbonate-irinotecan (5)

4-arm-PEG_(40k)-SCM (32.0 g) was dissolved in 400 mL CH₂Cl₂.2-(2-aminoethoxy)ethoxycarbonyl-irinotecan TFA salt (3) (2.85 g, 3.44mmol) was dissolved in 12 mL DMF and treated with 0.6 mL TEA, then addedto the solution of 4-arm-PEG_(40k)-SCM. The reaction was stirred at RTfor 12 hrs and then precipitated in Et₂O to get solid product, which wasdissolved in 1000 mL isopropyl alcohol (IPA) at 50° C. The solution wascooled to RT and the precipitate collected by filtration to give4-arm-PEG_(40k)-glycine-irinotecan (4) (drug content 3.7% based on HPLCanalysis). Yield: 59%.

Example 9 Formulation of 4-Arm-PEG-Gly-Irino-20K for IV Administration

4-arm-PEG-gly-irino-20K (as described in Example 1) was formulated as asterile lyophilized powder in lactate buffer in a pH range from 3 to 5.The product was packaged in sealed amber glass vials.

Prior to intravenous administration, the product is diluted with acommercially available dextrose solution, 5% w/w. The reconstituted drugsolution is diluted to a final concentration range of approximately 0.12to 2.8 mg/mL.

Example 10 In-Vitro Release of Irinotecan from 4-Arm-PEG-Gly-Irino-20K

The rate of release of irinotecan from 4-arm-PEG-gly-irino-20K wasexamined in vitro. Since the prodrug releases free irinotecan uponhydrolysis, its release rate is relevant to the pharmacokineticproperties of the molecule. Thus, a series of experiments was conductedto determine the impact, if any, of drug loading upon release kinetics.

Using a Type II dissolution tester coupled with HPLC analysis, thehydrolysis rate of irinotecan from 4-arm-PEG-gly-irino-20K in PBS at pH7.4 was assessed at 37° C.

The results are depicted in FIG. 14. Hydrolysis rate studies for twotypical product batches were run in triplicate over a period of 120hours. The resultant release profile was consistent and reproducible,and demonstrates a controlled release of irinotecan over the period ofstudy. The results further demonstrate that the hydrolysis rate isindependent of the average number of irinotecan molecules per moleculeof polymer, as well as the distribution of the irinotecan molecules onthe 4-arm PEG.

This finding was further supported by an additional study using a4-arm-PEG-gly-irino-20K composition that was synthesized to possess, onaverage, less than one molecule of irinotecan per 4-armed PEG (“low loadproduct”). The hydrolysis rate was then examined as described above, andthe results compared to a batch having the typical load of 2.3 to 3molecules of irinotecan per 4-armed polymer. The hydrolysis rate of the“low load product” was essentially the same as that shown in FIG. 14 fortypical product batches.

Example 11 Tumor Growth in Mice Implanted with MCF-7 Breast Tumor CellLine and Treated with 4-Arm-PEG-Gly-Irino-20K

Female athymic (Nu:Nu) mice were injected subcutaneously with MCF-7breast tumor cells and the tumors were allowed to reach a median volumeof 75 mm³. Each group of mice (n=10) was dosed every fourth day with atotal of 3 doses of 4-arm-PEG-gly-irino-20K at 20, 40, 60, or 90 mg/kgor irinotecan at 20 and 40 mg/kg. The control group received normalsaline. The animals were weighed and tumors were measured twice weeklyafter administration of the first drug injection.

All doses of 4-arm-PEG-gly-irino-20K (referred to simply as“PEG-irinotecan” in Table 2) and irinotecan were well tolerated with amaximum 10% loss in body weight. The effects of the test compounds ontumor regression are shown in Table 2 below.

TABLE 2 Summary of Tumor Growth Parameters for MCF-7 Tumor-Bearing MiceDays to Test Tumor Median 4 Times Com- Dose^(a) Regression^(b)Duration^(c) Tumor T − C^(e) pound (mg/kg) Partial Complete (days)Size^(d) (days) Control 0 0 0 NA 20.5 NA PEG- 90 1 0 49.0 >72 >52Irinotecan PEG- 60 0 0 NA >72 >52 Irinotecan PEG- 40 1 0 22.0 >72 >52Irinotecan PEG- 20 0 0 NA >72 >52 Irinotecan Irinotecan 40 1 0 42.0 42.822.3 Irinotecan 20 0 0 NA 40.2 19.7 ^(a)Refers to amount active compoundadministered in each dose. ^(b)Tumor regression: smallest tumor sizeafter the beginning of treatment relative to that observed on first dayof treatment. Partial: <50% of size observed on Day 1; Complete:unpalpable. ^(c)Interval during which partially or completely regressedtumor was below 50% pretreatment size. ^(d)Median number of days fortumor to quadruple in size from original volume. ^(e)Difference in themedian of times for tumors to gain 4 times original size for the druggroup minus that for the control group. NA: Not applicable

No tumor regrowth occurred in PEG-irinotecan-treated animals at any doselevel (FIG. 15A). The T-C value was significantly higher in thePEG-irinotecan treatment group than with the irinotecan treatment groupsat all 4 dose levels tested (p<0.001) showing significant growth delayin the PEG-irinotecan treatment groups compared to the irinotecantreatment groups.

For ease of comparison, FIG. 15C contains plots comparing mean tumorweight (mg) over time (days) in athymic mice implanted with MCF-7 humanbreast tumors following administration of 4-arm-PEG-gly-irino-20K,irinotecan, or a control. Subjects were administered 20 mg/kg irinotecanequivalent per dose; dosing occurred every four days for a total ofthree doses administered.

Example 12 Exposure (AUC) of 4-Arm-PEG-Gly-Irino-20K and its Metabolitesin a Colon Tumor Model

A PK/Pharmacodynamic (PD) study was conducted to evaluate whether agreater tumor suppression of 4-arm-PEG-gly-irino-20K over irinotecanobserved in a previous study could be explained by a greateraccumulation of SN38 in colon tumor tissue.

Fragments of human HT29 colon tumor were implanted subcutaneously infemale athymic mice. Tumors were allowed to reach approximately 170 mgin weight before the commencement of treatment. 4-arm-PEG-gly-irino-20Kor irinotecan were administered intravenously every four days for threedoses at a dose of 40 mg/kg. Pharmcokinetic sampling for plasma andtumor was at pre-selected time-points up to 60 days for both groups. Ateach time-point, four mice were sacrificed and blood and tumor sampleswere collected.

Pharmacokinetic parameters were estimated by compartmental PK analysesusing WinNonlin™ (professional version 2.1; Pharsight Corp., MountainView, Calif.), commercial software designed for the analysis of PK data.Computation of the AUC and the t½ in blood plasma and tumor tissue wasbased on estimation of primary kinetic parameters by least-squaresnonlinear regression routines for blood plasma and tumor tissueconcentration-time data.

TABLE 3 Exposure (AUC) of PEG-Irinotecan and its Metabolites in a ColonTumor Model Exposure (AUC) PEG-Irinotecan Irinotecan Ratio of (PEG-I)Treatment (I) Treatment PEG-I/I Analytes Plasma Tumor Plasma TumorPlasma Tumor SN38 5.8 11.7 0.01 0.03 531 366 Irinotecan 1.5 5.8 0.3 1.25 4.8 PEG- 2143 4598 NA NA NA NA Irinotecan PEG-SN38 784 2078 NA NA NANA Day 1 AUC of tumor tissue: days x μg/g Day 1 AUC of venous plasma:days x μg/mL Data obtained from nonlinear regression NA: Not applicable

AUC ratios of SN38 in plasma and tumor resulting from4-arm-PEG-gly-irino-20K administration were found to be of severalorders of magnitudes higher when compared to SN38 resulting fromirinotecan administration (Table 3). The tumor exposure to SN38following PEG-Irinotecan dosing was approximately 360-fold greater thanthat for following irinotecan dosing.

SN38 concentrations resulting from PEG-Irinotecan administrationdeclined at a much slower rate than with irinotecan administration(Table 4), yielding greater exposure following PEG-Irinotecanadministration. The t½ of SN38 measured following PEG-Irinotecanadministration in blood plasma and colon tumor tissue were 17 days and15 days, respectively. In contrast, SN38 resulting from irinotecanadministration resulted in more rapid blood plasma and intra-tumoral PK.This observed difference in SN38 PK appears to be the basis for greaterinhibition of tumor growth following PEG-Irinotecan administrationcompared to irinotecan in the in vivo colon tumor model investigated.

TABLE 4 Terminal Half-Life of PEG-Irinotecan and its Metabolitescompared to Irinotecan and SN38 in the Colon Tumor Xenograft ModelTerminal Half-life PEG-Irinotecan Irinotecan Treatment TreatmentAnalytes Plasma Tumor Plasma Tumor SN38 17 d 15 d 2 h 4 h Irinotecan  6h 10 d 1 h 2 h PEG-Irinotecan  4 h 15 d NA NA PEG-SN38 15 d 10 d NA NANLR: Nonlinear regression NA: Not applicable

Example 13 Pharmacokinetic Analysis of 4-Arm-PEG-Gly-Irino-20K and itsMetabolites Compared to Irinotecan in a Lung Tumor Model

A PK/PD study similar to that in Example 12 was conducted in a differenttumor model to evaluate whether the superior tumor suppression of4-arm-PEG-gly-irino-20K over irinotecan observed in a previous studycould be partially explained by an accumulation of SN38 in tumor tissue.Similar procedures and analyses were performed on mice implanted withNCI-H460 human lung tumor tissue as those described in Example 12.

TABLE 5 Exposure (AUC) of 4-arm-PEG-gly-irino-20K and its Metabolites ina Lung Tumor Model Exposure (AUC) PEG-Irinotecan Irinotecan Ratio of(PEG-I) Treatment (I) Treatment PEG-I/I Analytes Plasma Tumor PlasmaTumor Plasma Tumor SN38 2.6 10.5 0.2 0.18 11 59 Irinotecan 2.0 4.3 0.51.2 3.9 3.6 PEG- 1213 3555 NA NA NA NA Irinotecan PEG-SN38 229 1409 NANA NA NA Day 1 AUC of tumor tissue: days x μg/g Day 1 AUC of venousplasma: days x μg/mL NA: Not applicable

The tumor exposure to SN38 following PEG-Irinotecan dosing wasapproximately 60-fold greater than that following irinotecan dosing(Table 5). The t½ of SN38 in blood plasma and lung tumor tissuefollowing PEG-Irinotecan administration were 1 day and 6 days,respectively. In contrast, SN38 resulting from irinotecan administrationresulted in more rapid blood plasma and intra-tumoral PK (Table 6). Thisobserved difference in kinetics of SN38 appears to be the basis forgreater inhibition of tumor growth after PEG-Irinotecan administrationcompared to irinotecan in the in vivo lung tumor model.

TABLE 6 Terminal Half-Life of PEG-Irinotecan and its MetabolitesCompared to Irinotecan and SN38 in the Lung Tumor Xenograft ModelTerminal Half-Life PEG-Irinotecan Irinotecan Treatment TreatmentAnalytes Plasma Tumor Plasma Tumor SN38 1 d 6 d 2 h 15 h Irinotecan 10h  9 d 1 h  1 h PEG-Irinotecan 4 h 8 d NA NA PEG-SN38 2 d 14 d  NA NAData obtained from nonlinear regression NA: reliable t½ could not becalculated t½ is represented in hours and in those cases where the t½was greater than 24 hours, t½ is represented in days

The observed and predicted concentration time profiles of SN-38 in bloodplasma and in lung tumors, respectively, was plotted following repeatdosing with either PEG-irinotecan or irinotecan. SN38 resulting fromPEG-irinotecan administration demonstrated a monophasic decline in bloodplasma and in lung tumor tissue, as well as significant accumulation inboth. In contrast, minor accumulation of SN38 in tumor tissue wasobserved after dosing of irinotecan.

In summary, there was marked retention of SN38 in lung tumor in themouse xenograft model following PEG-Irinotecan dosing compared toirinotecan dosing. The changes were not as pronounced as those observedin the colon model. However, the favorable changes in SN38 kineticsfollowing PEG-Irinotecan dosing correlate well with superior suppressionof lung tumor growth demonstrated in this model.

Example 14 Synthesis of 4-Arm-PEG_(20k)-Glycine-Docetaxel: DMAP-DCCCoupling

4-Arm-PEG_(20K)-glycine (500 mg, 0.025 mmol) was dissolved in 10 mLmethylene chloride (DCM). 4-Dimethylaminopyridine (19 mg, 0.15 mmol) andDCC (32 mg, 0.15 mmol) were added to the PEG solution with stirring.After 5 minutes, docetaxel (121 mg, 0.15 mmol) was added and thereaction mixture continued to stir for an additional 24 h at roomtemperature. Upon completion, the reaction mixture was precipitated in amixed solvent system of ether/IPA (1:1). The resulting white solid wascollected by suction filtration, redissolved in DCM (2 mL) andreprecipitated using a single solvent system of diethyl ether (100 mL)to give the desired product after suction filtration.

¹H NMR analysis showed the existence of a significant amount ofN-acylurea (δ 1-2.5 ppm) byproduct. Subsequent drug release studies byHPLC revealed that about 40% of the PEG (one or more carboxylic groupsof each polymer molecule) was partially converted to the N-acylurea (acommon side product in a DMAP-DCC coupling reaction). Since theN-acylurea byproduct cannot be hydrolyzed back to the original PEGstarting material, the product purity profile becomes extremelycomplicated. Therefore, it is difficult to acquire accurate analysis,i.e. structural, drug loading, release rates, of the mixed productobtained using this method.

Example 15 Synthesis of 4-Arm-PEG20K-Glycine-Docetaxel

The overall synthesis of 4-arm PEG_(20K)-glycine docetaxel is shown inthe scheme above. The “4*” represents the theoretical number ofdocetaxel molecules per 4-armed polymer assuming complete drug loading.

A. Preparation of 4-arm-PEG_(20K)-glycine t-butyl ester

4-Arm-PEG_(20K)-CM (12.5 g, 0.625 mmol) was dissolved in 100 mL DCM.4-Dimethylaminopyridine (610 mg, 5.00 mmol) and DCC (625 mg, 3.00 mmol)were then added to the solution with stirring. After stirring for 5minutes, glycine t-butyl ester.HCl (503 mg, 3.00 mmol) was added and themixture continued to stir overnight at room temperature. Uponcompletion, the reaction mixture was precipitated using a mixed solventsystem of ether/IPA (1:1) to give the desired 4-arm-PEG_(20K)-glycinet-butyl ester product (10.5 g, 0.525 mmol, yield 84%) after suctionfiltration.

¹H NMR (CDCl₃) δ 4.11 (d, 8H), 4.05 (s, 8H), 3.90-3.37 (m, ˜1900H), 1.48(s, 36H).

B. Deprotection of 4-arm-PEG_(20K)-glycine t-butyl ester to form4-arm-PEG_(20K)-glycine

4-arm-PEG_(20K)-glycine t-butyl ester was deprotected usingtrifluoroacetic acid/methylene chloride (TFA/DCM, 3:1) and stirring atroom temperature for 3 h. The product was precipitated by addition ofether (600 mL) to the reaction mixture giving the desired4-arm-PEG_(20K)-glycine (9.2 g) after suction filtration.

¹H NMR (CDCl₃) δ 4.11 (d, 8H), 4.05 (s, 8H), 3.90-3.37 (m, ˜1900H).

C. Preparation of 4-arm-PEG_(20K)-glycine-docetaxel

DPTS was prepared as follows: p-Toluenesulfonic acid was dried byazeotropic distillation of a benzene solution, and then an equimolarsolution of DMAP in benzene was added. The resulting suspension wascooled to room temperature and the solid collected by suctionfiltration.

Docetaxel (776 mg, 0.96 mmol) and 4-arm-PEG_(20K)-glycine (4.0 g, 0.2mmol) were dissolved in 50 mL DCM, and then freshly prepared DPTS (155mg, 0.53 mmol), (Jeffrey S. Moore and Samuel I. Stupp, Macromolecules,1990, 23, 65-70) and DIC (404 mg, 3.2 mmol) were added with stirring.The reaction mixture continued to stir for 24 h at room temperature. Thereaction mixture was precipitated using a mixed solvent system ofether/IPA (1:1). The resulting white solid was collected by suctionfiltration, redissolved in 5 ml of DCM, and reprecipitated using asingle solvent system of ether (300 mL) to give the desired4-arm-PEG_(20K)-glycine-docetaxel after suction filtration. Yield: 90%.

¹H NMR (CDCl₃) δ 8.12 (d, 8H), 7.73 (m, 4H), 7.61 (m, 4H), 7.52 (m, 8H),7.41 (m, 8H), 7.33 (m, 8H), 6.20 (t, 4H), 5.69 (m, 8H), 5.60 (m, 4H),5.36 (s, 4H), 5.22 (m, 4H), 4.97 (d, 4H), 4.33 (m, 8H), 4.30 (m, 12H),4.06 (d, 8H), 3.98 (s, 8H), 3.90-3.24 (m, ˜1900H), 2.60 (m, 4H), 2.36(m, 20H), 1.96 (s, 12H), 1.86 (m, 8H), 1.75 (s, 12H), 1.68 (m, 8H), 1.35(s, 36H), 1.25 (s, 12H), 1.13 (s, 12H). All chemical shift values in ppm(5).

D. Drug Loading and Hydrolysis of 4-arm-PEG_(20K)-glycine-docetaxel

Drug loading was determined ¹H NMR (8%) and RP-HPLC (6.2%) analyticalmethods while hydrolysis rates (in phosphate buffer) were determinedexclusively by RP-HPLC.

Calculation of the Drug Loading by ¹H NMR:

Samples of different PEG-docetaxel concentrations were prepared, and thenumber of scans was then varied depending on the concentration of thesample. Based on the averaged proton peak integration of all spectraobtained, the drug loading was determined.

Calculation of the Drug Loading and Hydrolysis Rate by HPLC

Instrument: HP 1100

Column: C₁₈ column

Mobile Phase: A: 0.1% TFA in H₂O; B: Acetonitrile

Flow Rate: 0.5 mL/min

Gradient Table:

Time (min) A % B % 0 60% 40% 15 0 90%

Drug Loading Determination:

Drug loading and hydrolysis rates were determined experimentally asfollows: Using the abovementioned HPLC method, 10.8 mg docetaxel wasdissolved in a mixed solvent system of acetonitrile/PBS (1:1, 10 mL, pH7.4). This stock solution was further diluted serially to give thefollowing concentrations of docetaxel solutions: 540 μg/mL, 405 μg/mL,300 μg/mL, 216 μg/mL, 108 μg/mL and 54 μg/mL. The peak areas wereobtained for each concentration and a standard curve was generated. Then30.4 mg of 4-arm-PEG_(20K)-docetaxel was dissolved in 10 mL PBS, pH 7.4.The solution was filtered, and then aliquots of 0.3 mL were placed into10 individual HPLC vials. These vials were stored at 37° C. and prior touse, 0.3 mL of acetonitrile was added to ensure all PEG-docetaxel andfree docetaxel present in the sample were dissolved. One vial was usedfor each injection, and injections were made at various timepoints overa course of 200 h (8.3d). The appearance of free drug released from thePEG-conjugate was monitored and upon completion the final concentrationwas determined against the standard curve.

The drug loading value refers to the average number of docetaxelmolecules covalently attached to the polymer in the4-arm-PEG_(20K)-glycine-docetaxel product. The calculated molecularweight of the 4-arm PEG_(20K)-glycine-docetaxel product, assuming 4docetaxel molecules per 4-arm polymer, is approximately 23,232. Themolecular weight of docetaxel is 808. Assuming complete drug loading (4docetaxels per polymer), the theoretical percent weight of drugcontained in the product is (3232/23232)100 or 13.9%. The actualobserved weight of drug, as determined by HPLC, was 6.2%, whichcorresponds to an average number of docetaxels per polymer of 1.78. Thedrug loading value determined by ¹H NMR was 8%, which corresponds to anaverage number of docetaxel molecules per polymer of about 2.3. Thus,for this preparation, based upon the average of both methods, theaverage number of docetaxel molecules per polymer is slightly higherthan 2.00.

Half-Life Determination.

The determination of hydrolysis rate, reported as a half-life, utilizedthe same analytical method outlined above for the drug loading. Once thedrug release was complete, the half-life was calculated by eitherdetermining the time at which the concentration (area %) of the freedrug equaled 50% or, if perfectly linear, by determining the slope ofthe plot (In 1-S % vs. hour) and using the following equation:

Half-life=In(2)/slope

The half-life for 4-arm-PEG_(20K)-Docetaxel was determined to be 15.3 h.

Example 16 Anti-Tumor Activity of 4-Arm-PEG_(20K)-Glycine-Docetaxel inMice Implanted with NCI-H460 Lung Tumors

Human NCI-H460 lung tumors (30 to 40 fragments of each) were implantedsubcutaneously in the mice (Charles Rivers Labs: NCr nu/nu) near theright axillary area. The day of implantation was designated Day 0 andthe tumors were allowed to reach a weight of 100-245 mg in weight priorto treatment.

The animals were randomized into groups in a manner such that the mediantumor weights on the first day of treatment were as close to each otheras possible.

Treatment:

The mice received 1 or 2 intravenous doses of test compound or vehicle(saline).

Tumor Measurement:

The animals were weighed and the tumors measured twice weekly afteradministration of the first injection. The tumor volume was measured bycaliper measurements (mm) and using the formula of an ellipsoid sphere:L×W²/2=mm³, where L and W refer to the larger and smaller perpendiculardimensions collected at each measurement. This formula was also used tocalculate tumor weight assuming unit density (1 mm³=1 mg).

Study Duration:

Any animal found moribund or any animal whose tumor reached 4000 mg,ulcerated or was sloughed off was euthanized prior to study termination.

Results:

Two different efficacy studies were conducted. The 1^(st) studyevaluated the efficacy of 4-arm-PEG_(20K)-Docetaxel and docetaxelagainst H460 NSCLC tumors. FIGS. 16A-C illustrate the effect of a singledose of each compound on the tumor growth. It was observed that doses of20 and 40 mg/kg of the PEGylated docetaxel provided an improvedanti-tumor effect over the un-PEGylated free compound. The 10 mg/kg doseshowed a significant difference between the two compounds.

In the 2^(nd) study, the anti-tumor efficacy (H460 NSCLC tumors) wasmeasured up to the maximum tolerated dose for athymic nude mice. Theanimals tolerated docetaxel up to 30 mg/kg and PEG-docetaxel up to 60mg/kg. FIG. 17 illustrates the effect of two doses (q7d×2) of eachcompound on the tumor growth. It is again evident from the results, thatthe PEGylated compound provided an improved anti-tumor effect over theDocetaxel compound. A dose response is clearly evident among the threePEGylated drug doses when compared to the three un-PEGylated drug doses.

Example 17 Anti-Tumor Activity of 4-Arm-PEG_(20K)-Glycine-Docetaxel inMice Implanted with DU-145 Prostate Tumors

The study was carried out as described in Example 16 above, with theexception that the tumors used were DU-145 prostate tumors.

The anti-tumor efficacy was evaluated against prostate tumors (DU-145)up to the maximum tolerated dose of each compound. The animals tolerateddocetaxel up to 30 mg/kg and PEG-docetaxel up to 60 mg/kg.

FIG. 18 illustrates the anti-tumor effect of two doses (q7d×2) of eachcompound. It is again evident from the results that the PEGylatedcompounds completely suppressed the tumor growth at all 3 doses testedand for the 78 day observation period. The docetaxel compound showedgood activity, but the tumors did recover and grow after 30-50 days.

Example 18 Anti-Tumor Activity of 4-Arm-PEG_(20K)-Glycine-Docetaxel inMice Implanted with MCF-7 Breast Tumors

Up to 100 mice (Charles Rivers Labs: CD-1 Fox nl nu) were surgicallyimplanted in the lateral side of the neck with a subcutaneous17β-estradiol (estrogen) pellet (1.00 mg/pellet; Innovative Research ofAmerica, Sarasota, Fla., USA) at least 2 days prior to cell inoculation.These pellets release estrogen at a rate of 0.011 mg/day for 90 daysafter implant. Following surgery, approximately 1×10⁶ MCF-7 cells in avolume of 0.1 mL phosphate buffered saline (PBS)/Matrigel™ (1:1 v/v)were injected subcutaneously in the right flank. The tumors were allowedto reach a range of 50-150 mm³. Day 0 for this study corresponded to thefirst day of dosing.

The animals were randomized into groups in a manner such that the mediantumor weights on the first day of treatment were as close to each otheras possible.

Treatment:

The mice received 1 or 2 intravenous doses of test compound or vehicle(saline).

Tumor Measurement:

The animals were weighed and the tumors measured twice weekly afteradministration of the first injection. The tumor volume was measured bycaliper measurements (mm) and using the formula of an ellipsoid sphere:L×W²/2=mm³, where L and W refer to the larger and smaller perpendiculardimensions collected at each measurement. This formula was also used tocalculate tumor weight assuming unit density (1 mm³=1 mg).

Study Duration:

Any animal found moribund or any animal whose tumor reached 1500 cc,ulcerated or was sloughed off was euthanized prior to study termination.

Results:

The anti-tumor efficacy was evaluated against breast tumors (MCF-7) atdoses of 10, 20 and 30 mg/kg. The results showed complete suppression oftumor growth at the two high doses and for both compounds tested. FIG.19 illustrates the anti-tumor effect of the 10 mg/kg dose (q7d×2).

Example 19 Treatment of Advanced Colorectal Cancer in Irinotecan-NaïveSubjects with 4-Arm-PEG-Gly-Irino-20K and Combination with Cetuximab

The study population consists of patients having advanced colorectalcancer, and who have failed one prior therapy. The subjects are alsoirinotecan-naïve. The study is an open-label, randomized, double armstudy designed to evaluate 4-arm-PEG-gly-irino-20K as a second linetreatment for colorectal cancer in irinotecan naïve subjects.

Subjects are administered either 4-arm-PEG-gly-irino-20K at a startingdose of 100 mg/m² up to 175 mg/m² (irinotecan equivalents) or irinotecanweekly, in combination with cetuximab. Cetuximab, also administered onceweekly, is administered at an initial loading dose of 400 mg/m² on Day 1as a two hour intravenous infusion, and then continued weekly at a doseof 250 mg/m² administered as a one hour intravenous infusion. Theprimary endpoint of the study is progression-free survival.

Example 20 Additional Cytotoxicity Studies with 4-Arm-PEG-Gly-Irino-20Kand 4-Arm-PEG20K-Glycine-Docetaxel

Additional illustrative in-vivo studies were carried out as describedpreviously herein or in accordance with methods well-known to one havingordinary skill in the art. The results of such studies are providedgraphically herein as FIGS. 20-26.

These results provide additional evidence for the superior efficacy ofthe prodrugs provided herein in preclinical mouse xenograft models ofmultiple tumor types. These studies indicate superior tumor regressionaccompanied by reduced toxicities in rats and dogs for the prodrugsprovided herein when compared to unmodified drug alone. Moreover, basedupon the results illustrated, for example, in FIG. 20, administration of4-arm-PEG-gly-irino-20K results in a two to three times logarithmicincrease in exposure to SN-38 in colorectal tumors in mice in comparisonto irinotecan.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art.

Example 21

Antitumor Activity of 4-Arm-PEG-Gly-Irino-20K when Administered as aSingle Agent and in Combination with an Anti-Angiogenic Agent in a HumanColorectal Tumor Xenograft Model

HT29 tumor fragments were implanted into female, athymic nude mice inthe monotherapy study. Following establishment of measurable tumors ingroups of 10 mice, a total of three irinotecan equivalent dosesadministered every fourth day (q4d×3) of 4-arm-PEG-gly-irino-20K oririnotecan (40, 60, or 90 mg/kg). Animals were weighed and tumor volumesmeasured twice weekly after the first drug injection in both studies.

For the combination study, athymic nude female mice were inoculatedsubcutaneously with HT29 tumor cells. Once measurable tumors wereestablished, the mice were dosed on Day 1 with either 20 or 40 mg/kg4-arm-PEG-gly-irino-20K (irinotecan equivalents), and/or 50 μg/doseAvastin. 4-arm-PEG-gly-irino-20K was administered IV every seven daysfor a total of three irinotecan equivalent doses (q7d×3) and Avastin wasadministered IP on Days 1 and 14. Efficacy was evaluated by determiningthe in-study tumor growth inhibition (TGI).

Significant antitumor activity was observed in all mice receiving4-arm-PEG-gly-irino-20K as a monotherapy compared to irinotecan. Tumorgrowth delay was significantly longer with 4-arm-PEG-gly-irino-20K atall three doses tested (20.9-60.2 days) compared with irinotecan(0.3-1.6 days). 1/10 animals achieved partial tumor regression, and 3/10animals achieved complete tumor regression at the 90 mg/kg dose. Alldoses of 4-arm-PEG-gly-irino-20K and irinotecan were well tolerated.

20 mg/kg 4-arm-PEG-gly-irino-20K+Avastin resulted in 31% and 30%increases in TGI compared to 4-arm-PEG-gly-irino-20K and Avastin alone,respectively. There were 2/10 partial tumor regressions in thiscombination group. 40 mg/kg 4-arm-PEG-gly-irino-20K+Avastin resulted in5% and 46% increases in TGI compared to 4-arm-PEG-gly-irino-20K andAvastin alone, respectively. There were 8/10 partial regressions and onecomplete regression within this combination group. The combinationregimens were well-tolerated with no signs of overt toxicity.

4-arm-PEG-gly-irino-20K inhibited tumor growth more effectively thanirinotecan in the HT29 human colorectal tumor xenograft model.4-arm-PEG-gly-irino-20K in combination with Avastin demonstrated greateranti-tumor activity than when administered as a monotherapy in thismodel.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

What is claimed is:
 1. A method of treating cancer in a mammaliansubject, said method comprising: administering a therapeuticallyeffective amount of a composition comprising a prodrug having thestructure:

where n ranges from 40 to 500, to a subject having one or more canceroussolid tumors, over a duration of time effective to produce an inhibitionof growth of said one or more cancerous solid tumors in said subject. 2.The method of claim 1, wherein said cancerous solid tumor type isselected from the group consisting of colorectal, ovarian, breast,non-small cell lung and prostate.
 3. The method of claim 2, wherein thecancer is breast cancer.
 4. The method of claim 2, wherein the cancer iscolorectal cancer.
 5. The method of claim 2, wherein the cancer isnon-small cell lung cancer.
 6. The method of claim 2, wherein the canceris prostate cancer.
 7. The method of claim 2, wherein the cancer isovarian cancer.
 8. The method of claim 1, wherein the mammalian subjectis human.
 9. The method of claim 1, wherein the overall nominal averagemolecular weight of the prodrug ranges from about 20,000 to about 80,000daltons.
 10. The method of claim 1, wherein the composition furthercomprises one or more additional prodrugs having a structure selectedfrom the group consisting of:

wherein O-Irinotecan is

L is the site of attachment, and n ranges from 40 to
 500. 11. The methodof claim 1, wherein the composition further comprises a pharmaceuticallyacceptable carrier.
 12. The method of claim 1, wherein saidadministering is parenteral.
 13. The method of claim 12, wherein saidparenteral administering is via a route selected from intraperitoneal,intravenous, subcutaneous, and intramuscular injection.
 14. The methodof claim 13, wherein said administering is by intravenous infusion. 15.The method of claim 14, wherein the composition comprises a sterileaqueous preparation of the prodrug.
 16. The method of claim 7, whereinthe dosage administered comprises from about 10.0 to about 60 mgirinotecan/kg body weight of the subject.